JP2002530105A - Methods for Cloning Pigs - Google Patents

Abstract

  (57) [Summary] The present invention relates to materials and methods for cloning pigs. The present invention relates, in part, to totipotent cells useful for cloning porcine animals, porcine embryos produced from such cells by using nuclear transfer techniques, and porcine animals resulting from such cells and embryos. About.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to the cloning of pigs.

BACKGROUND OF THE INVENTION [0002] The following description of the background of the invention is to be understood as merely providing an aid to the reader for understanding the invention and as describing or constituting prior art to the invention. is not.

[0003] Researchers have developed methods for cloning mammals over the past two decades. Some of the methods reported include (1) isolating cells, usually embryonic cells;
) Inserting the cell or a nucleus isolated from the cell into an enucleated oocyte (eg, the nucleus of the oocyte has been previously removed) and (3) allowing the embryo to mature in vivo; Process.

[0004] The success of the first nuclear transfer experiment using mammalian cells was reported in 1983, where pronuclei isolated from mouse zygotes were inserted into enucleated oocytes. Got a living offspring. McGrath and Solter, 1983, Science 220: 1300-13
02. Another then described the generation of chimeric mouse embryos using mouse primordial germ cells (PGCs), for example, embryos containing a subset of cells with nuclear DNA that is significantly different from other cells in the embryo. did. These cells are pluripotent cells and can give rise to pluripotent cells. Matsui et al., 1992, Cell 70: 841-847, and Resnick
Et al., 1992, Nature 359: 550; Kato et al., 1994, Journal of Reproduction and Fert.
ility, Abstracts, Fertility Research Society, Annual Meeting, Southampton, 13:38, 1998. Researchers use mouse hill cells as nuclear donors in cloning techniques to establish cloned mouse family animals. We reported what we could do. Wakayama et al.
1998, Nature 394: 369-374.

[0005] Another nuclear transfer experiment was reported in 1986, where cloned sheep were born, using sheep embryonic cells as nuclear donors in the cloning process. More recently, another sheep was reported to have been cloned from sheep embryonic cells; serum-depleted somatic cells; cells isolated from the scutellum; and somatic cells of the breast. Campbell et al., 1996, Na
Nature 380: 64-66; PCT Publication WO 95/20042; Wilmut et al., 1997, Nature 3.
85: 810-813; and PCT publications WO 96/07732 and WO 97/0766.
9. Other approaches to cloning sheep have involved manipulating the activation state of in vivo matured oocytes following nuclear transfer. PCT Publication WO 97/07668. Publications disclosing cloned sheep report cloning efficiencies of up to about 0.4%. The cloning efficiency calculated for the above estimation is a ratio equal to the number of sheep cloned divided by the number of nuclear transpositions used to generate that number of cloned sheep.

[0006] Yet another nuclear transfer experiment yielded a cloned bovine animal in which the animal was cloned using embryonic cells derived from 2-64 cell embryos as nuclear donors. This bovine animal is disclosed in US Pat.
The clone was reported to have been cloned using the nuclear transfer technique disclosed in US Pat. Others reported that cloned bovine embryos were generated using the inner cell mass cells of a blastocyst stage embryo as a nuclear donor for nuclear transfer. Sims and First
1993, Theriogenology 39: 313 and Keefer et al., 1994, Mol.Reprod.Dev. 38:26.
4-268. In addition, another publication reported that cloned bovine embryos were produced by nuclear transfer technology utilizing PGCs isolated from fetal tissue as nuclear donors. Delhaise et al., 1995, Reprod. Fert. Develop. 7: 1217-1219; Lavoir 1994.
37: 413-424; and a PCT entitled "Embryonic Stem Cell-Like Cells", J. Reprod.
Application WO 95/10599.

[0007] With respect to pigs, researchers have reported a method for obtaining chimeric animals, specifically a method of placing a nuclear donor inside enucleated embryo cells. Prather et al., 198
9, Biology of Reproduction 41: 414-418; Piedrahita et al., 1998, Biology of Re
production 58: 1321-1329; and WO 94/26884, "Embryonic stem cells for producing chimeric and transgenic ungulates", Wheeler, published November 24, 1994.

In addition, researchers have reported nuclear transfer experiments for porcine nuclear donors and porcine oocytes. For example, Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; N
agashima et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod.
41: 414-419; Prather et al., 1990, Exp. Zool. 255: 355-358; Saito et al., 1992, Assis.
.Reprod.Tech.Andro. 259: 257-266; and Terlouw et al., 1992, Theriogenology 3.
See 7: 309.

In addition, researchers have reported methods for activating porcine oocytes. Grocholova
Et al., 1997, J. Exp. Zoology 277: 49-56; Schoenbeck et al., 1993, Theriogenology 40.
: 257-266; Prather et al., 1991, Molecular Reproduction and Development 28: 405
-409; Jolliff and Prather, 1997, Biol. Reprod. 56: 544-548; Mattioli et al., 1
991, Molecular Reproduction and Development 30: 109-125; Terlouw et al., 1992
Fri., Theriogenology 37: 309; Prochazka et al., 1992, J. Reprod. Fert. 96: 725-734; F.
unahashi et al., 1993, Molecular Reproduction and Development 36: 361-367; Pra
Ther et al., Bio.Rep. 50 Vol. 1: 282; Nussbaum et al., 1995, Molecular Reproduction.
and Development 41: 70-75; Funahashi et al., 1995, Zygote 3: 273-281; Wang et al.,
1997, Biology of Reproduction 56: 1376-1382; Piedrahita et al., 1989, Biology
of Reproduction 58: 1321-1329; Machaty et al., 1997, Biology of Reproduction 5
7: 85-91; and Machaty et al., 1995, Biology of Reproduction 52: 753-758.

[0010] There remains a long-felt need for materials and methods that produce effective nuclear transfer using porcine nuclear donors. This long pressing need is partly due to the potential medical applications known as xenotransplantation, which include methods for removing organs from pig animals and transplanting the organs into humans in need thereof. Is based on U.S. Patent No. 5,589,582, Hawley et al., Registered December 31, 1991; PCT application WO.
95/28412, Baetsher et al., Published October 26, 1995; PCT application WO.
96/06165, Sachs et al., Published February 29, 1996; PCT application WO93.
/ 16729, Bazin, published September 2, 1993; PCT application WO 97/120.
35, Diamond et al., Published April 3, 1997; PCT application WO 98/16630.
Piedrahita and Bazer, published April 23, 1998.

SUMMARY The present invention relates, in part, to cloning techniques for porcine animals. The invention further provides, in part, totipotent cells and cells that can be made totipotent for use in cloning methods and in the generation of porcine animals, produced from the cells of these pigs using nuclear transfer techniques. Embryos, porcine animals born from these cells and embryos, and methods and processes for establishing such cells, embryos, and animals.

The present invention provides a number of advantages over currently available means and methods for cloning pigs. Such features and advantages include: (1) Production of cloned porcine animals from virtually any type of cell. The present invention provides materials and methods for reprogramming non-totipotent porcine cells into totipotent porcine cells. These non-totipotent porcine cells may be of non-embryonic origin. This feature of the invention allows for the phenotype of an existing porcine animal to be assessed and then a totipotent cell line for cloning the animal to be easily established. (2) Establishment of a totipotent porcine cell line from virtually any type of porcine cells. In one embodiment of the invention, non-totipotent porcine progenitor cells can be reprogrammed into totipotent cells. These non-totipotent progenitor cells may be non-embryonic cells. Established totipotent porcine cell lines offer the advantage of increased cloning efficiency due to low heterogeneity inside the cell line (eg, cell lines often show lower differentiation rates than primary cells). Also, totipotent cell lines can be engineered in vitro to generate porcine cells, embryos, and genome-engineered animals (eg, transgenics). In addition, totipotent cell lines can be more easily stored, transported, and re-established in culture than other types of cell lines. (3) Enhanced efficiency of embryo cloning as a result of utilizing an asynchronous and karyotypically stable porcine cell line in a complete in vitro embryo production system.

[0013] Cloning efficiency can be expressed by the ratio of the number of embryos resulting from nuclear transfer to the number of nuclear transfers performed to produce that embryo. Alternatively, cloning efficiency can be expressed as the ratio of the number of live birth animals to the number of nuclear transpositions performed to produce these animals.

Cultured Cells According to the Invention In a first aspect, the invention features totipotent porcine cells. As used herein, the term "pig" can refer to any animal of the family Suidae. Pigs can refer to any type of pig including, but not limited to, wild boar, domestic pigs, miniature pigs, warthog, peccary, and barboosa. For examples of minipigs, see, e.g., Bustad and McClellan, 1968, Lab.Anim.Care. 18: 280-287 and England and Panepinto, 1986, "Conceptual and operational history of the development of miniature pigs", Swine in Biomedical Research. (Edited by METubleson), Plenum Press
, NY 17-22, all of which are hereby incorporated by reference in their entirety, including all drawings, tables and drawings.

The term “totipotent” as used herein can refer to cells that give rise to live birth animals. The term "totipotent" can further refer to cells that give rise to all cells in a particular animal. Totipotent cells can give rise to whole cells of an animal when used in a method of generating embryos from one or more nuclear transfer steps. Totipotent cells are also used to produce defective animals, such as those useful for harvesting organs, e.g., animals that have a genetic modification that eliminates organ or appendage growth by manipulating homeogenes. be able to.

As used herein, the term “live birth” preferably refers to animals that exist outside the uterus. A "live birth" animal may be an animal that has been alive for at least one second since it left the maternal host. "Live birth" animals may not require the circulatory system of the intrauterine environment for survival. A "live birth" animal may be a walkable animal. Such animals can include prepubertal and postpubertal animals. As mentioned above, live birth animals may lack some of what is present in normal animals of that type.

In a preferred embodiment, the totipotent cells are (1) cultured; (2) cultured as a cell line; and (3) cultured as a permanent cell line.

As used herein, the term “cultured” as used with reference to a cell can refer to one or more cells that undergo or do not undergo cell division in an in vitro environment. The in vitro environment can be any medium known in the art that is suitable for maintaining cells in vitro, such as a suitable liquid medium or agar. A specific example of a suitable in vitro environment for cell culture is the Culture of Anima
l Cells: a manual of basic techniques (3rd edition), 1994, RIFreshney (ed.), W
iley-Liss, Inc .; Cells: a laboratory manual (1 volume), 1998, DLSpector, RD
Goldman, LA Leinwand (eds.), Cold Spring Harbor Laboratory Press; and An
imal Cells: culture and media, 1994, DC Darling, SJ Morgan John Wiley a
nd Sons, Ltd., each of which is incorporated herein by reference in its entirety, including all drawings, tables and drawings. The cells are suspended and / or
Or, in a monolayer, it can be cultured with one or more substantially similar cells. Cells can be cultured in suspension and / or monolayer with heterogeneous cell populations. The term "heterogeneous" as used in the preamble may relate to any cellular property, such as, for example, cell type and cell cycle. Cells can be cultured in suspension, cultured as a monolayer attached to a solid support, and / or cultured, for example, on a layer of feeder cells. The term "feeder cells" is defined below. further,
Cells can be successfully cultured by plating under conditions that lack cell-to-cell contact. Preferably, the cultured cells undergo cell division and have at least 5
Culturing for more days, more preferably for at least 10 or 20 days, most preferably for at least 30 days. Preferably, a significant number of the cultured cells do not end in culture. "End" and "substantial number" will be defined later. Almost any type of cell can be placed in cell culture conditions. Cultured cells can be used to establish cell lines.

The term “cell line,” as used herein, can refer to a cultured cell that can be passaged without terminating at least once. The present invention provides at least 1,2,5,1
It relates to cell lines that can be passaged 0, 15, 20, 30, 40, 50, 60, 80, 100 and 200 times. Cell passage is defined below.

As used herein, the terms “terminate” and “terminate” as used in reference to cultured cells refer to a number of techniques known to those of skill in the art, such as the CytoTox96 ™ cytotoxicity assay, Promega, Inc., catalog No. G1780; Celltiter96 ™ aqueous cell proliferation assay kit, Promega, Inc., catalog no. G3580; and trypan blue solution for cytotoxicity assays, Sigma, catalog no. It may mean a cell that undergoes death. Termination can also be accomplished using a number of techniques known to those of skill in the art, such as the Dead End ™ Apoptosis Detection Kit, Promeg
a, Inc., catalog no. G7130), which can be the result of apoptosis, which can be measured. Terminated cells are identified as cells that have undergone cell death and / or apoptosis and have been released from the solid surface of the culture. In addition, the terminated cells may lack intact membranes that can be identified by the methods described above. Also, terminated cells may show a decrease in metabolic activity, which may be caused in part by a decrease in mitochondrial activity that can be identified, for example, by rhodamines 1,2,3. In addition, termination can mean cell culture in which a significant number of cultured cells have been terminated. The term "substantial number" in the preceding sentence refers to about 8 of the cells in culture.
0%, preferably about 90% of the cells in culture, more preferably about 1% of the cells in culture.
00%, and most preferably 100% of the cells in culture.

As used herein, the term “suspension” can refer to a cell culture condition in which the cells are not attached to a solid support. Cells growing in suspension,
Stirring can be performed using equipment well known to those skilled in the art.

The term “monolayer” as used herein can refer to cells that are attached to a solid support while growing in a suitable culture. Under appropriate growth conditions, a small portion of the cells growing in the monolayer may be attached to the cells in the monolayer, but not to the solid support. Preferably less than 15% of these cells are not attached to a solid support, more preferably less than 10% of these cells are not attached to a solid support, and most preferably these cells Less than 5% are not attached to the solid support.

As used herein, the term “substantially similar” as used with respect to porcine cells can refer to cells from the same organism and the same tissue. The term "substantially similar" can further refer to a cell population that has not significantly differentiated. For example, preferably less than 15% of the cells in the cell population are differentiating, more preferably more than 10% of the cell population.
Less than 5% of the cells are differentiated, most preferably less than 5% of the cell population is differentiated.

As used herein, the term “plated” or “plated” as used with reference to cells may mean establishing a cell culture in vitro. For example, cells can be diluted in a cell culture medium and then added to a cell culture plate, dish, or flask. Cell culture plates are widely known to those skilled in the art. Cells can be plated at various concentrations and / or cell densities.

The term “cell plating” can also be expanded to the term “cell passage”. The cells according to the present invention can be passaged using cell culture techniques well known to those skilled in the art. The term "passaging cells" refers to (1) releasing the cells from a solid support or substrate and dissociating the cells, and (2) diluting the cells in a medium suitable for further cell growth. Can be pointed out. Cell passage further removes a part of the liquid medium containing the cultured cells,
It can also mean adding a liquid medium to the original culture to dilute the cells and allow further cell growth. In addition, cells can be added to a new culture vessel containing a medium suitable for further cell growth.

As used herein, the term “proliferation” as used in reference to a cell can refer to a group of cells that can increase in number over a period of time.

As used herein, the term “confluent” can refer to a group of cells in which a majority of the cells are in physical contact with at least one other cell in the group. Confluence can also be defined as a group of cells growing to a maximum cell density under given conditions. For example, if a group of cells can grow in a monolayer and they are placed in a culture vessel with a suitable growth medium,
As the monolayers spread over a significant surface area of the culture vessel, they become dense. The surface area converted by the cells is preferably about 50% of the total surface area, more preferably about 70% of the total surface area, and most preferably about 90% of the total surface area.

As used herein, the term “permanent” or “immortalized” as used with respect to porcine cells, refers to the number of cells undergoing cell division during culture in an in vitro environment, It can refer to cells that can be doubled. Permanent cell lines can be doubled ten times before a significant number of cells are terminated in culture. Preferably, the permanent cell line can be doubled over 20 or 30 times before a significant number of cells are terminated in culture. More preferably, the permanent cell line can be doubled, forty or fifty times, before a significant number of cells are terminated in culture. Most preferably, the permanent cell line can be doubled over 60 times before a significant number of cells are terminated in culture. The term "quit" was mentioned earlier. Cell doubling can be measured by counting the number of cells in culture using techniques well known to those skilled in the art. As a measure of cell culture permanence, the number of doublings until a significant number of cells are terminated in culture can be measured. The term "substantial number" was also mentioned before.

[0029] Permanent cells can be distinguished from non-permanent cells on the basis that they can be passaged at a lower density than non-permanent cells. In particular, permanent cells can be grown to confluency (discussed below) if the plating conditions do not allow physical contact between the cells. Thus, permanent cells can be distinguished from non-permanent cells when the cells are plated at a cell density at which the cells do not physically contact each other.

In a preferred embodiment, (1) the totipotent cells are not alkaline phosphatase positive (eg, the cells are not appreciably stained for alkaline phosphatase); (2) the totipotent cells arise from at least one progenitor cell (3) progenitor cells are isolated and / or produced from any region of the porcine animal; (4) progenitor cells are isolated and / or produced from any cells in culture; , Non-embryonic cells, non-embryonic cells, differentiated cells, undifferentiated cells, somatic cells, embryonic cells, fetal cells, embryonic stem cells, primordial germ cells, germ cells, hill cells, amniotic cells, fetal fibroblasts, hepatocytes, embryonic germ cells, adult cells, cells isolated from the asynchronous cell population, and, the tuning population is not stopped by the G ₀ phase of the cell cycle, synchronously cells Cells from Dan isolated, selected from the group consisting of; (6) totipotent cells have a morphology of embryonic germ cells.

As used herein, the term “alkaline phosphatase positive” can refer to the presence of detectable alkaline phosphatase in a cell. Cells that are not alkaline phosphatase positive do not stain appreciably using methods to visualize cellular alkaline phosphatase. Methods for detecting the presence of alkaline phosphatase in a cell are well known to those skilled in the art. For example, Matsui et al., 1991, "Effects of Steel factor and leukemia inhibitory factor on mouse primordial germ cells in culture," Nature.
353: 750-752. Examples of cells that stain appreciably alkaline phosphatase can be found in the literature. See, for example, Hogan's U.S. Pat. The entire contents including tables and drawings are part of this specification.

As used herein, the term “progenitor cell” or “progenitor cell group” can refer to a cultured porcine cell or a cell or group of cells used to establish a cultured porcine cell line. . The progenitor cell or group of cells can be isolated from almost any cell entity. For example, a progenitor cell or group of cells may be a blastocyst, embryo, fetus,
And can be isolated from cell lines (eg, cell lines established from embryonic cells), preferably from fetal and / or cell lines established from fetal cells, and more preferably from extrauterine animals And / or from cell cultures and / or cell lines established from such extra-uterine animals. Extrauterine animals include neonatal animals (eg, 5 days after birth), adolescent animals (eg, prepubertal animals), adolescent animals (eg, after ovulation or production of live sperm), and adult animals (eg, postpubertal). Can exist as An extrauterine animal can be a living or post-mortem animal. The progenitor cells may or may not be cultured.
In addition, progenitor cells can be cryopreserved or frozen at the same time (eg, cryopreserved cells can be used as progenitor cells to establish cell culture).
. These examples are not intended to be limiting and a further description of these exemplary progenitor cells is provided below.

As used herein, the term “derived from” means that one or more cells
It can refer to converting to one or more cells having at least one different property. For example, (1) non-totipotent progenitor cells can be converted to totipotent cells by utilizing the features of the present invention described below; (3) progenitor cells can give rise to cultured cells; (4) progenitor cells can give rise to cultured cell lines;
(5) Progenitor cells can give rise to cultured progenitor cell lines. The conversion process can be referred to as a reprogramming step. In addition, the term "derived from" can refer to establishing a totipotent embryo from a totipotent cell according to the present invention by using the nuclear transfer process described below.

As used herein, the term “reprogramming” or “reprogrammed” can refer to materials and methods that can convert a cell into another cell having at least one different property. . Also, such materials and methods can reprogram or convert a cell to another cell type that is not typically expressed during the life cycle of the cell. For example, (1) non-totipotent cells can be reprogrammed into totipotent cells; (2) progenitor cells can be reprogrammed into cells with EG cell morphology; and ( 3) Progenitor cells can be reprogrammed into totipotent cells. Examples of materials and methods for converting progenitor cells into totipotent cells having the morphology of EG cells are described below.

As used herein, the term “isolated” can refer to a cell that has been mechanically separated from another population of cells. Examples of groups of cells are developing cell masses, cell cultures, cell lines, and animals. These examples are not intended to be limiting, and the invention relates to any population of cells. Methods for isolating one or more cells from another group of cells are well known in the art. For example, Culture of
Animal Cells: a manual of basic techniques (3rd edition), 1994, RIFreshney (
Ed.), Wiley-Liss, Inc .; Cells: Research Manual (1), 1998, DLSpector, R.
D. Goldman, LA Leinwand (eds.), Cold Spring Harbor Laboratory Press; and
Animal Cells; culture and media, 1994, DC Darling, SJ Morgan, John Wil
See ey and Sons, Ltd.

[0036] As used herein, the term "non-embryonic cell" can refer to a cell that is not isolated from an embryo. Non-embryonic cells may or may not be differentiated. Non-embryonic cells can refer to almost any somatic cell, for example cells isolated from an extrauterine animal. These examples are not intended to be limiting.

For the purposes of the present invention, the terms “embryo” or “embryonic” as used herein
It can refer to a growing cell mass that is not implanted in the uterine membrane of the maternal host. Thus, the term “embryo” as used herein refers to an inseminated oocyte, a cybrid (as defined herein), a developing cell mass at the preblastocyst stage, and / or It can refer to other developing cell masses that are in a developmental stage before implantation into the endometrium of the host. Embryos according to the invention may not display reproductive ridges. Thus, “embryonic cells” are isolated from and / or arise from an embryo.

An embryo can represent multiple stages of cell development. For example, a one-cell embryo can be called a zygote, a solid cell globule resulting from a cleaved embryo can be called a morula, and an embryo with a blastocoel can be called a blastocyst. Can be.

As used herein, the term “fetal” can refer to a growing cell mass that has implanted within the endometrium of a maternal host. The fetus may include distinct features such as, for example, a reproductive ridge. Reproductive ridges are features that are easily identified by those skilled in the art, and that are recognizable in fetuses of most animal species. As used herein, the term “fetal cell” can refer to any cell isolated from and / or derived from or derived from a fetus, and includes amniotic cells. The term "non-fetal cell" is a cell that is not derived or isolated from a fetus.

When the progenitor cells are isolated from a fetus, such progenitor cells are preferably isolated from a porcine fetus, wherein the fetus is between 20 and parturition, between 30 and 100 days, more preferably Is a fetus between days 35 and 70 and between days 40 and 60, and most preferably about 55 days. Fetal age can be determined by the time that the embryo that develops into the fetus is established. For example, a two-cell embryo is referred to as a one-day embryo, which can develop into a 54-day embryo. The term "about" with respect to a fetus can refer to plus or minus 5 days.

As used herein, the term “delivery” can refer to the time at which a fetus is delivered from a female recipient. The fetus is delivered from a female recipient by miscarriage, cesarean section, or birth.

[0042] As used herein, the term "primordial germ cell" can refer to a diploid progenitor cell that can become a germ cell. Primordial germ cells can be isolated from any tissue in the developing cell mass, and are preferably isolated from germ-raised cells of the developing cell mass. Reproductive ridges are part of a growing cell mass that is well known to those skilled in the art. See, for example, Strelchenko, 1996, Theriogenology 45: 130-141 and Lavoir 1994, J. Reprod. Dev. 37: 413-424.

As used herein, the term “embryonic stem cells” can refer to pluripotent cells isolated from an embryo maintained in in vitro cell culture. Embryonic stem cells can be cultured with or without feeder cells. Embryonic stem cells can be established from embryonic cells isolated from embryos at any developmental stage, including blastocyst stage embryos and preblastocyst stage embryos. Embryonic stem cells have a round cell morphology and can grow into round cell clumps on a support layer. Embryonic stem cells are well known to those skilled in the art. See, for example, WO97 / 37009 (published October 9, 1997) by Stice and Goluke entitled "Culture of Inner Cell Lines Derived from Ungulate Embryos," and Yan
g and Anderson, 1992, Theriogenology 38: 315-335, each of which is hereby incorporated by reference in its entirety, including all drawings, tables, and drawings. See, for example, Piedrahita et al., 1998, Biol. Reprod. 58: 1321-1329; Wia.
nny et al., 1997, Biol. Reprod. 57: 756-764; Moore and Piedrahita, 1997, In Vi.
tro Cell Biol. Anim. 33: 62-71; Moore and Piedrahita, 1996, Mol. Reprod. De.
v. 45: 139-144; Wheeler, 1994, Reprod. Fert. Dev. 6: 563-568; Hochereau-de R
eviers and Perreau, Reprod. Nutr. Dev. 33: 475-493; Strojek et al., 1990, Theri.
See Piogenrahita et al., 1990, Theriogenology 34: 879-901; and Evans et al., 1990, Theriogenology 33: 125-129, each of which is incorporated by reference in all drawings, tables, and drawings. The entire content including is incorporated herein by reference.

As used herein, the term “differentiated cell” can refer to a precursor cell that has developed from a non-specific phenotype to a specialized phenotype. For example, embryonic cells can differentiate into epithelial cells that line the intestinal tract. The materials and methods according to the present invention can reprogram differentiated cells into totipotent cells. Differentiated cells can be isolated, for example, from fetal or live birth animals.

As used herein, the term “undifferentiated cell” can refer to a precursor cell that has a non-specific phenotype and is capable of differentiating. An example of an undifferentiated cell is a stem cell.

As used herein, the term “asynchronous population” can refer to cells that have not arrested at any stage of the cell cycle. While many cells can progress along the cell cycle and do not stay at any one stage, some cells can become at one stage of the cell cycle for some period of time. Some known stages of the cell cycle are G ₁ , S, G ₂ and M. The asynchronous population of cells is not manipulated to tune to any one of these phases, or predominantly to any one of these phases. By utilizing a number of techniques known in the art, such as, for example, colcemid exposure, cells can be stopped at the M phase of the cell cycle. An example of a method for arresting cells at one stage of the cell cycle is discussed in WO 97/07669, entitled "Hibernate Cell Population for Nuclear Transfer," which is incorporated by reference in all figures, The entire contents including tables and drawings are part of this specification.

As used herein, the terms “synchronized population” and “synchronized” can refer to a portion of cells within a population that are within the same stage of the cell cycle. Preferably, about 50% of the cells in the cell population remain in one stage of the cell cycle, more preferably, about 70% of the cells remain in one stage of the cell cycle, and most preferably About 90% of the cells in a cell population remain in one stage of the cell cycle. The stages of the cell cycle can be distinguished by the relative size of the cells and by various cell markers known by those skilled in the art. For example, cells can be identified with such markers using flow cytometry techniques well known to those skilled in the art. Alternatively, cells can be identified by size using techniques well known to those skilled in the art, for example, by use of light microscopy and micrometer. In a preferred embodiment, the cells are synchronized by remaining at certain discrete stages of the cell cycle (ie, the cells do not divide).

As used herein, the terms “embryonic germ cells” and “EG cells” are cultured cells having a distinct flattened morphology, which can grow in culture in a monolayer. EG cells can be distinguished from fibroblasts. This EG cell morphology is in contrast to cells that have a rounded morphology and form multiple aggregates on the support layer.
Porcine embryonic germ cells may not require the presence of a feeder layer or the presence of growth factors in cell culture conditions. Porcine embryonic germ cells further proliferate at a reduced doubling rate as these cells approach confluence on culture plates. The porcine embryonic germ cells according to the invention can be totipotent. The porcine embryonic germ cells according to the invention may not stain appreciably for alkaline phosphatase. Preferably, the porcine embryonic germ cells are established in a medium containing a significant concentration of glucose, as described herein.

[0049] Porcine embryonic germ cells can be established from cell cultures of almost any type of porcine progenitor cells. Examples of progenitor cells are described herein, and a preferred progenitor cell for establishing a porcine embryonic germ cell culture is a fetal germ cell. The reproductive ridge cells are preferably isolated from porcine fetuses, wherein the fetuses are between 20 and parturition, between 30 and 100 days, more preferably between 35 and 70 days and between 40 and 60 days. And most preferably about 55 days old fetus. Fetal age can be determined as described above. The term "about" with respect to a fetus can refer to plus or minus 5 days. As described herein, EG cells can be physically isolated from a primary culture of cells, and the isolated EG cells are used to establish a cell culture that is ultimately homogenous or nearly homogenous. To form a cell line of a suitable EG cell.

As used herein, the terms “morphology” and “cell morphology” refer to cell morphology,
It can refer to structure, and physical properties. For example, certain cell morphologies are at a significant level of alkaline phosphatase, and the cell morphology can be identified by determining whether the cells stain recognizablely for alkaline phosphatase. Another example of cell morphology is whether the cells are flat or round when cultured on the surface or in the presence of a feeder cell layer. Many other cell morphologies are known to those of skill in the art, and cell morphologies are readily identifiable using materials and methods that are well known to those of skill in the art. For example, Culture of Animal Cells: a manual o
See f basic techniques (third edition), 1994, RIFreshney (eds.), Wiley-Liss, Inc.

As used herein, the term “hill cell” refers to the tissue surrounding an oocyte and / or
Or it can refer to any cultured or uncultured cells isolated from the cells. Those skilled in the art can easily identify cumulus cells. Examples of methods for isolating and culturing cumulus cells are described in Damiani et al., 1996, Mol. Reprod. Dev. 45: 521-
534; Long et al., 1994, J. Reprod. Fert. 102: 361-369; and Wakayama et al., 1998, N.
394: 369-373, each of which is incorporated herein by reference in its entirety, including all drawings, tables, and drawings.

The term “amniotic cells” as used herein can refer to cultured or uncultured cells isolated from amniotic fluid or tissue in contact with amniotic fluid. One skilled in the art can easily identify amniotic cells. Examples of methods for isolating and culturing amniotic cells are described in Bellow et al., 1996, Theriogenology 45: 225; Garcia and Salaheddine, 1997,
Theriogenology 47: 1003-1008; Leibo and Rail, 1990, Theriogenology 33:53.
1-552; and Vos et al., 1990, Vet. Rec. 127: 502-504, each of which is hereby incorporated by reference in its entirety, including all drawings, tables, and drawings. Part of

As used herein, the term “fetal fibroblast” can refer to any differentiated porcine fetal cell having the appearance of a fibroblast. Fibroblasts can have a flattened appearance when cultured on culture media plates. Fetal fibroblasts also have a spindle-like morphology, a limited density for proliferation, and can have a finite culture life of approximately 50 generations. In addition, fetal fibroblasts maintain a tight diploid chromosome content and can produce type I collagen. For a description of fibroblasts,
For example, Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994
And RIFreshney (eds.), Wiley-Liss, Inc.

[0054] As used herein, the term "adult cell" can refer to any cell isolated from an adult porcine animal. Such adult cells can be isolated from any portion of pig animals, including but not limited to ear-derived skin, abdominal skin, kidney, liver, heart, and lung. is not. Methods for culturing such adult cells are disclosed herein.

[0055] In a preferred embodiment, the totipotent porcine cells according to the invention comprise modified nuclear DNA; (2) the modified nuclear DNA comprises a recombinant product-encoding DN.
(3) the recombinant product is a polypeptide; (4) the recombinant product is a ribozyme; (4) the recombinant product is expressed in a biological fluid or tissue; (5) the recombinant product confers resistance to one or more diseases;
Or (6) the recombinant product confers or partially confers resistance to one or more parasites; and (7) the modified nuclear DNA functions as a regulatory element. (8) the regulatory element is selected from the group consisting of a promoter, enhancer, insulator, and repressor; and (9) the regulatory element is a milk protein promoter, urine. Protein promoter, blood protein promoter, lacrimal protein promoter, synovial fluid protein promoter, mandibular gland protein promoter, casein promoter, β-casein promoter, melanocortin promoter, milk serum protein promoter, α-lactalbumin promoter, whey acid protein promoter, uro Plakin promoter,
It is selected from the group consisting of the α-actin promoter.

As used herein, the term “modified nuclear DNA” refers to a cell, embryo, fetus, or animal nuclear deoxyribonucleic acid according to the present invention, which has been manipulated by one or more recombinant DNA techniques. Can refer to an array. Examples of recombinant DNA technology are well known to those skilled in the art and include (1) inserting a DNA sequence from another organism (eg, a human) into the target nuclear DNA; Or higher DN
Removing the A sequence and (3) introducing one or more base mutations (eg, site-directed mutations) into the target nuclear DNA. Cells with altered nuclear DNA are, for purposes of the present invention, "transgenic cells".
Can be referred to as Transgenic cells can be useful as material for the nuclear transfer cloning techniques described herein.

Methods and devices for the insertion, removal and mutation of nuclear DNA in mammalian cells are well known to those skilled in the art. Molecular Cloning, a Laboratory Manual, 2nd
Edition, 1989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laborato
ry Press; U.S. Patent No. 5,633,067, "Method for Generating Transgenic Bovine or Transgenic Bovine Embryo", DeBoer et al. (registered May 27, 1997); U.S. Patent No. 5,612,205, "Homologous recombination of mammalian cells", Kay et al. (1997 3
And PCT Publication WO 93/22432, "Methods for Identifying Transgenic Preimplantation Embryos"; WO 98/16630, Piedrahita and Bazer, 199.
See “Methods for the Generation of Primordial Germ Cells and Transgenic Animal Species”, published April 23, 8, each of which, by reference, includes the entire contents, including all drawings, drawings, and tables. Is part of the present specification. These methods are based on outpatient D
Techniques for transfecting cells with NA fragments and proper design of foreign DNA fragments, such that the foreign DNA fragment performs insertion, removal, and / or mutation of the target DNA genome. .

[0058] Transgenic cells can be obtained in various ways. For example, transgenic cells can be isolated from a transgenic animal. Examples of transgenic pig animals are well known in the art. Cells isolated from a transgenic animal can be converted to totipotent cells by using the materials and methods described herein. In another example, transgenic cells can be established from totipotent cells according to the invention. Materials and methods for converting non-transgenic cells to transgenic cells are known in the art, as described above.

[0059] Any of the cell types defined herein can be altered to have altered nuclear DNA. For example, embryonic stem cells, embryonic germ cells, fetal cells, and any totipotent cells as defined herein, have modified nuclear DNA.
Can be changed.

Examples of methods for altering the target DNA genome by insertion, removal, and / or mutation include retroviral insertion, artificial chromosome technology, gene insertion, random insertion using a tissue-specific promoter, homologous recombination, Gene targeting, transposable elements,
And / or any other method for introducing foreign DNA. Other modification techniques well known to those skilled in the art include removal of DNA sequences from the genome and / or
Or alteration of the nuclear DNA sequence. Examples of techniques for altering nuclear DNA sequences include:
It is a method of site-directed mutagenesis and polymerase chain reaction. Thus, the invention relates, in part, to porcine cells that are simultaneously totipotent and transgenic. Such transgenic and totipotent cells may serve as an almost unlimited source of donor cells for producing cloned transgenic pig animals.

As used herein, the term “recombinant product” can refer to a product generated from a DNA sequence that includes at least a portion of a modified nuclear DNA. This product can be, for example, a peptide, polypeptide, protein, enzyme, antibody, antibody fragment, polypeptide bound to a regulatory element (terms described below), structural protein,
It may be an RNA molecule, and / or a ribozyme. These products are well-defined in the art. This listing of products is for illustrative purposes only, and the invention also relates to other types of products.

[0062] As used herein, the term "ribozyme" can refer to a ribonucleic acid molecule capable of cleaving another RNA molecule at a specific region. Ribozymes are RN
It can bind to separate regions on the A molecule and then specifically cleave regions within or adjacent to the binding region. This allows ribozyme technology
The amount of polypeptide translated from previously intact message RNA molecules can be reduced. For a detailed description of ribozymes, see Cech et al., US Pat.
54855 (registered October 11, 1994), and "RNA ribozyme polymerase, dephosphorylase, restriction endoribonucleases and methods".
See U.S. Pat. No. 5,591,610 (registered Jan. 7, 1997) to Cech et al.
Each of these is incorporated herein by reference in its entirety, including all drawings, tables, and drawings.

The term “biological fluid” or “tissue” as used herein can refer to any fluid or tissue in a living organism. Fluids include, but are not limited to, tears, saliva, milk, urine, amniotic fluid, semen, plasma, tubal fluid, and synovial fluid. Tissues include lung, heart, blood, liver, muscle, brain, pancreas, skin, etc.,
It is not limited to these.

As used herein, the term “conferring resistance” can refer to the ability of a recombinant product to completely eliminate or partially reduce the symptoms or parasitic state of a disease. . Thus, if a disease is associated with inflammation, for example, if the inflammation is reduced upon expression of the recombinant product, the recombinant product can confer resistance to the inflammation. The recombinant product confers resistance to a disease or parasitic condition, or is a partial sequence, if it is an antisense RNA that specifically binds to an mRNA molecule encoding a polypeptide that contributes to inflammation. Can be provided. Other examples of conferring disease or parasite resistance are described below. In addition, examples of diseases are described below.

Examples of parasites and strategies for conferring resistance to these parasites are described below. Examples of these include, but are not limited to, helminths, nematodes, insects, invertebrates, bacteria, viruses, and eukaryotic parasites. These parasites can lead to disease states that can be controlled by the materials and methods of the present invention.

As used herein, the term “modulator” can refer to a DNA sequence that can increase or decrease the amount of product produced from another DNA sequence. Regulatory elements can result in constitutive production of the product (eg, the product can be constantly expressed). Alternatively, a regulatory element can inducibly enhance or decrease the production of a recombinant product (eg, the product can be expressed in response to a specific signal). Regulatory elements can be controlled, for example, by nutrition, by light, or by adding certain substances to the system of the transgenic organism. Examples of regulatory elements well known to those skilled in the art are promoters, enhancers, insulators, and repressors. For example, Transgenic Animals, Generation and
See Use, 1997, ed. LM Houdebine, Hardwood Acedemic Publishers, Australia, which is hereby incorporated by reference in its entirety, including all drawings, tables, and drawings.

As used herein, the term “promoter group” or “promoter”
It can refer to a DNA sequence located adjacent to the DNA sequence encoding the recombinant product. The promoter is preferably operably linked to an adjacent DNA sequence. Promoters typically increase the amount of recombinant product expressed from a DNA sequence relative to the amount of recombinant product expressed in the absence of the promoter. DNA derived from another species using a promoter derived from one species
Expression of the recombinant product from the sequence can be enhanced. In addition, one promoter element can increase the amount of recombinant product expressed for multiple tandemly linked DNA sequences. Thus, one promoter element can enhance the expression of one or more recombinant products. Numerous promoter elements are well known to those skilled in the art. Examples of promoter elements are described below.

As used herein, the term “enhancer group” or “enhancer”
It can refer to a DNA sequence located adjacent to the DNA sequence encoding the recombinant product. An enhancer element can typically be located upstream of a promoter element or downstream of an encoding DNA sequence (eg, a DNA sequence that is transcribed or translated into the recombinant product (s)). Thus, the enhancer element is a 100 base pair, a 200 base pair, of the DNA sequence encoding the recombinant product.
Alternatively, it can be located upstream by 300 or more base pairs. The enhancer element has a higher D value than the increased expression provided by the promoter element.
The amount of recombinant product expressed from the NA sequence can be increased. Numerous enhancer elements are readily available to one skilled in the art.

As used herein, the term “insulators” or “insulator” can refer to a DNA sequence that is adjacent to a DNA sequence encoding a recombinant product. Insulator elements can direct the expression of recombinant products to specific tissues of the organism. Numerous insulator elements are well known to those skilled in the art. See, for example, Geyer, 1997, Curr. Opin. Genet. Dev. 7: 242-248,
This is hereby incorporated by reference in its entirety, including all drawings, tables and drawings.

As used herein, the term “repressor” or “repressor element” can refer to a DNA sequence that is located near a DNA sequence that encodes a recombinant product. Presser sequences can reduce the amount of recombinant product expressed from the DNA sequence. The repressor element can be controlled by attaching a specific molecule or specific molecules to the repressor element DNA sequence. These molecules can either activate or inactivate the repressor element. Numerous repressor elements are available to those skilled in the art.

The terms “milk protein promoter”, “urine protein promoter”, “blood protein promoter”, “laminary duct protein promoter”, “synovial protein promoter”, and “mandibular gland protein promoter” have been specifically identified in animals. Refers to a promoter element that regulates the specific expression of a protein within a given fluid or gland or cell type. For example, a milk protein promoter is a regulatory element capable of controlling the expression of a protein expressed in animal milk. Other promoters are well known to those skilled in the art, for example, the casein promoter, the α-lactalbumin promoter, the whey protein promoter, the uroplakin promoter, and the α-actin promoter.

In a preferred embodiment, (1) the totipotent porcine cell has undergone an operation; (2) the operation comprises utilizing the totipotent porcine cell in a nuclear transfer method; (3) the operation is a totipotent cell. (4) the step of thawing totipotent cells; (5) the step of subculturing totipotent cells; (6) the step of totipotent cells (7) the operation comprises replacing the totipotent cells with foreign DN.
Transfection with A; and (8) the step of dissociating the cells from another cell or group of cells.

As used herein, the term “operation” can refer to the general usage of the term management or handling for some purpose. Examples of operations are described herein.

As used herein, the term “nuclear transfer” refers to a cell that has been enucleated from a cell.
It may mean introducing the whole of the nuclear DNA. Nuclear transfer methods are well known to those skilled in the art. For example, Nagashima et al., 1997, Mol. Reprod. Dev. 48: 339-343; Nagashima
Et al., 1992, J. Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41: 414-4.
19; Prather et al., 1990, Exp. Zool. 255: 355-358; Saito et al., 1992, Assis. Reprod.
259: 257-266; and Terlouw et al., 1992, Theriogenology 37: 309, each of which is hereby incorporated by reference in its entirety, including all drawings, tables, and drawings. Part of Nuclear transfer can be achieved using oocytes that are not surrounded by zona pellucida.

As used herein, the term “cold storage” can refer to freezing a cell, embryo, or animal according to the present invention. The cells, embryos or parts of the animals according to the invention are preferably frozen at a temperature below 0C, more preferably below -80C, and most preferably below -196C. The cells and embryos according to the present invention can be cryopreserved indefinitely. It is known that biological materials can be stored cold for more than 50 years and still survive. For example, cows can be artificially inseminated using bovine semen that has been cryopreserved for more than 50 years, resulting in the birth of living offspring. Cryopreservation methods and equipment are well known to those skilled in the art. For example, Voelkel U.S. Pat. No. 5,160,031, entitled "Cryopreservation Process for Direct Transfer of Embryos"
No. 2 (registered November 3, 1992).

The term “thaw” as used herein can refer to the process of raising the temperature of a cryopreserved cell, embryo, or portion of an animal. Methods of melting cryopreserved materials so that they are active after the melting process are well known to those skilled in the art.

As used herein, the terms “transfected” and “transfection” refer to a method of bringing exogenous DNA into a cell. These methods involve treating cells with, for example, high concentrations of salts, electric fields, liposomes, polycationic micelles, or detergents to make the outer membrane or wall of the host cell permeable to the nucleic acid molecule of interest. And various other technologies. These detailed methods are not limiting and the invention relates to any transformation technique well known to those skilled in the art. For example, Molecular Cloning, a Laboratory Manual, 2nd edition, 1
989, Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Pr
See, ess, and Transgenic Animals, Generation and Use, 1997, ed. LM Houdebine, Hardwood Acedemic Publishers, Australia, both of which were previously incorporated herein by reference.

As used herein, the term “foreign DNA” can refer to DNA that can be transfected into a target cell, where the foreign DNA is nuclear DNA of the target organism
Has at least one base pair of modification. Foreign DNA and transfection can be further understood and defined in connection with the term "modified nuclear DNA" mentioned above.

As used herein, the term “dissociate” can refer to materials and methods useful for separating cells from other cells, where the cells are originally in contact with each other. For example, blastomeres (ie, cell members of the morula embryo) can be detached from the rest of the growing cell mass by techniques and equipment well known to those skilled in the art. For example, US Pat. No. 4,994,384 (199) entitled "Bovine Embryo Propagation"
(Registered Feb. 19, 2001), which is hereby incorporated by reference in its entirety, including all drawings, tables and drawings. Alternatively, cells growing in culture can be separated from one another to facilitate processes such as the passage and formation of EG cells described herein. Further, the departure of one cultured cell from a group of cultured cells can be useful as a first step in the process of nuclear transfer described below. When cells are dissociated from embryos, the dissociation can be useful for processes such as recloning, the processes described herein, as well as steps for amplifying some embryos.

In another embodiment, the present invention provides a method comprising: (a) isolating at least one progenitor cell; and (b) culturing the progenitor cell in a cell culture medium, wherein at least one in the culture medium. The cell develops embryonic germ cell morphology]. In a preferred embodiment, the process comprises the steps of: (1) introducing a stimulus to the progenitor cells that converts the progenitor cells into totipotent porcine cells; and (2) the process comprising a cell comprising a substantial concentration of at least one carbohydrate. Culturing progenitor cells in the culture medium; (3) the carbohydrate is glucose; and (4) the cell culture medium contains one or more antibiotics.

The term “convert” as used herein can refer to the phenomenon by which progenitor cells become totipotent. The term “convert” is synonymous with the term “reprogram” as used herein when the progenitor cells are non-totipotent cells. Progenitor cells can be converted to totipotent cells at various rates. For example, it is possible to convert only a small portion of progenitor cells to totipotent cells.

The term “stimulation” as used herein can refer to materials and / or methods useful for converting progenitor cells into totipotent cells. The stimulus can be, for example, electrical, mechanical, temperature-related, and / or chemical. The stimuli may be a combination of one or more different types of stimuli. The stimulus can be introduced to the progenitor cells at any time that conversion of the progenitor cells to totipotent cells can be achieved.

As used herein, the term “introducing” as used in connection with a stimulus can refer to the step or steps of contacting the progenitor cells with the stimulus. If the nature of the stimulus is, for example, chemical, the stimulus can be introduced into progenitor cells by mixing the stimulus with a cell culture medium.

As used herein, the term “substantial concentration of at least one carbohydrate” can refer to a cell culture medium that contains at least one carbohydrate at a concentration that does not lyse or shrink the cultured cells. Cultured cells can lyse or shrink when the osmotic pressure of the culture medium is too high. Cells can be grown over a wide range of osmolarity (eg, 260 mOsm / k
g and 320 mOsm / kg) can be tolerated. Increasing the carbohydrate concentration in the medium dramatically increases the osmotic pressure of the medium, which can affect cell viability. For example, Cells: a laboratory manual (1 volume), 1998, DLSpector, RDGold
See man, LA Leinwand (eds.), Cold Spring Harbor Laboratory Press, which is hereby incorporated by reference in its entirety, including all drawings, tables, and drawings.

[0085] The carbohydrate can be any monosaccharide, disaccharide, or polysaccharide known in the art. Examples of carbohydrates are glucose, mannose, dextrose, mannose, idose, galactose, talose, growth, altrose, allose, ribose, arabinose, xylose, lyxose, threose, erythrose, glyceraldehyde, sucrose, lactose, maltose, cellulose. ,
And glycogen, but are not limited thereto. A particularly preferred carbohydrate is glucose. The preferred glucose concentration in the cell culture medium is 1
More than mM glucose, more preferably more than 5 mM glucose, 10 mM glucose, and 15 mM glucose, and most preferably about 17 mM glucose. The term "about," as used in reference to glucose concentration, can refer to plus or minus 2 mM glucose.

[0086] As used herein, the term "antibiotic" can refer to any molecule that reduces the rate of growth of bacteria, yeast, fungi, molds, or other contaminants in cell culture. Antibiotics are optional components of cell culture media. Examples of antibiotics are well known in the art. See Sigma and DIFCO catalogs.

In a preferred embodiment, (1) progenitor cells are cultured with feeder cells; (2) progenitor cells are not cultured with feeder cells; (3) feeder cells are established from fetal cells; (4)
Fetal cells arise from a fetus in which no cell type has been removed from the fetus (eg, dissociate the entire fetus into a cell culture system); (5) The fetal cells may have one or more cell types. Originating from a fetus that has been removed from the fetus (eg, removing the head region and detaching the rest of the fetus into a cell culture system); (6) introducing a stimulus into the progenitor cells by supporting cells; (7) Feeder cells are the only source of stimulation; (
8) the stimulus is introduced into the precursor cells in a mechanical manner; (9) the only stimulus introduced into the precursor cells is introduced in the mechanical manner; (10) the stimulus is introduced into the precursor cells by the feeder cells in a mechanical manner. (11) stimulating comprises incubating the progenitor cells with the mixture of receptor ligands; (12) isolating the progenitor cells from an ungulate, preferably a porcine animal; , Non-fetal cells, differentiated cells, undifferentiated cells, somatic cells, embryonic cells, fetal cells, embryonic stem cells, primordial germ cells, reproductive ridge cells, cumulus cells, amniotic cells, fetal fibroblasts, hepatocytes , embryonic germ cells, adult cells, asynchronous cell populations isolated from cells, and synchronously from a cell population isolated cells [wherein, synchronously population stopped in G ₀ phase of the cell cycle Absent];
(14) The receptor ligand mixture is at least one selected from the group consisting of cytokines, growth factors, trophic factors, and neurotrophic factors, LIF, and FGF.
(15) LIF has an amino acid sequence substantially similar to the amino acid sequence of human LIF; and (16) FGF has an amino acid sequence substantially similar to that of bovine bFGF.

As used herein, the terms “mechanical modality” and “mechanical stimulus” can refer to introducing a stimulus into a cell, where the stimulus is not introduced by a supporting cell. For example, purified LIF and bFGF (defined below)
These purified products can be introduced as a stimulus to progenitor cells by adding them to a cell culture medium in which the progenitor cells are growing. Again, as described herein, significant amounts of glucose can be added to the culture medium as a stimulus to the cells.

[0089] As used herein, the term "feeder cells" can refer to cells that are maintained in culture and cultured with target cells. Target cells can be, for example, progenitor cells, embryonic stem cells, embryonic germ cells, cultured cells, and totipotent cells. Supporting cells include, for example, peptides, polypeptides, electrical signals, organic molecules (eg, steroids)
, Nucleic acid molecules, growth factors (eg, bFGF), other factors (eg, cytokines such as LIF and Steel factor), and metabolic nutrients for target cells. Certain cells, such as embryonic germ cells, cultured cells, and totipotent cells, may not require feeder cells for healthy growth. The feeder cells are preferably grown in a monolayer.

[0090] Feeder cells can be established from a number of cell types. Examples of these cell types are fetal cells,
Mouse cells, buffalo rat hepatocytes, and fallopian tube cells. These examples are not intended to be limiting. Tissue samples can be disrupted to establish feeder cell lines by methods well known in the art (eg, by using a blender). The feeder cells can be from the same or different animal species as the progenitor cells. Feeder cells can be established from ungulate fetal cells, porcine fetal cells, and mouse fetal cells. Removing one or more cell types from the fetus (
The support layer can be established from, for example, primordial germ cells, cells in the head region, and cells in the body cavity region), from these removed cells, or from the remaining fetal cells from which the limbs have been cut. When utilizing an entire fetus to establish fetal support cells, the feeder cells (eg, fibroblasts) and progenitor cells (eg, primordial germ cells) can originate from the same source (eg, a single fetus).

The term “mixture of receptor ligands” as used herein can refer to a mixture of one or more receptor ligands. A receptor ligand can mean any molecule that binds to a receptor protein located outside or inside a cell. Receptor ligands can be selected from cytokine family ligands, neurotrophic factor family ligands, growth factor family ligands, and mitogen family ligand molecules, all of which are well known to those skilled in the art. Examples of receptor / ligand pairs are: epidermal growth factor receptor / epidermal growth factor, insulin receptor / insulin, cAMP-dependent protein kinase / cAMP, growth hormone receptor / growth hormone, and steroid receptor / steroid. Certain receptors have been shown to exhibit cross-reactivity. For example, heterologous receptors such as insulin-like growth factor 1
(IGFR1) and insulin-like growth factor receptor 2 (IGFR2) both bind to IGF1. If the receptor-ligand mixture contains a stimulus, the receptor-ligand mixture can be introduced into the progenitor cells in various ways known to those skilled in the art.

[0092] The term "cytokine" as used herein can refer to a large family of receptor ligands that are well known to those of skill in the art. Receptor ligands of the cytokine family include leukemia inhibitory factor (LIF); cardiotrophin 1
(CT-1); reticular neurotrophic factor (CNTF); stem cell factor (SCF), also known as steel factor; oncostatin M (OSM); and IL-6
, IL-11, and any member of the interleukin (IL) family, including IL-12, and the like. The teachings of the present invention do not require the mechanical addition of Steel factor (also known in the art as stem cell factor) for the conversion of progenitor cells to totipotent cells.

[0093] As used herein, the term "growth factor" refers to any receptor ligand that can elicit a cell growth effect, elicit a cell growth effect, and / or affect cell morphology. Can be. Examples of growth factors are well known in the art.
Fibroblast growth factor (FGF) is an example of a growth factor. The term "bFGF" can mean basic FGF.

As used herein, the term “substantially similar” as used with respect to amino acid sequences preferably refers to 50% or more amino acid matches, more preferably 70% or more amino acid matches, or most preferably It can refer to two amino acid sequences having 90% or more amino acid identity. Amino acid matches are properties of amino acid sequences that evaluate their similarity or relatedness. Matches are evaluated by dividing the number of matching residues in the two sequences by the total number of residues and multiplying the result by 100. Thus, two copies with exactly the same sequence are 100
Sequences that have% matches, are less highly conserved, and have deletions, additions, or substitutions have a lower degree of matches. One skilled in the art will appreciate that several computer programs are available for performing sequence comparisons and determining sequence identity.

It has been discovered that when culturing progenitor cells in vitro, cells having a different cell morphology from the progenitor cells can be generated without introducing a stimulus to the progenitor cells. For example, a progenitor ridge cell can be referred to as a supporting cell,
It has been found that they can develop into cells with EG cell morphology without contacting with receptor ligands or growth factors. Thus, in a preferred embodiment, (1) progenitor cells are not contacted with exogenous receptor ligand; (2) progenitor cells are not contacted with exogenous growth factor; (3) progenitor cells are not contacted with supporting cells; 4
) Progenitor cells are not contacted with exogenous receptor ligand and are not contacted with exogenous growth factor; (5) progenitor cells are not contacted with exogenous receptor ligand and are not contacted with supporting cells; Cells are not contacted with exogenous growth factor and do not come into contact with supporting cells; and (7) progenitor cells are not contacted with exogenous receptor ligand, and are not in contact with exogenous growth factor, and contact with supporting cells Do not let.

As used herein, the term “exogenous” as used in reference to a growth factor or receptor ligand refers to the exogenous form of the receptor ligand and / or growth factor that can be added to a substrate or medium that contacts the target cell. Can refer to a source. For example, purified bFGF, which is commercially available to those skilled in the art, can be added to a cell culture medium that contacts the progenitor cells. In this latter example, such purified bFGF can be referred to as "exogenous bFGF". A number of exogenous receptor ligands and / or a number of exogenous growth factors or combinations thereof can be added to the liquid medium containing the cells. Alternatively, as described above, the progenitor cells may not need to be contacted with exogenous growth factors or exogenous receptor ligands.

In another embodiment, the invention features a method of producing a totipotent porcine cell comprising the steps of: (a) isolating one or more progenitor cells; and
b) A stimulus is introduced into the progenitor cells that converts the progenitor cells into totipotent cells. Any of the embodiments defined herein above for totipotent porcine cells relate to a method for producing totipotent porcine cells. In yet another aspect, the invention features a method of producing a totipotent porcine cell comprising the steps of: (a) isolating at least one progenitor cell; and (b) isolating the progenitor cell. The totipotent cells are established by culturing in cell culture medium, wherein the totipotent cells have the form of embryonic germ cells.

Cloned Embryos According to the Invention The present invention relates, in part, to cloned totipotent porcine embryos.
Thus, embodiments of the present invention include (1) embryos that are totipotent; (2) embryos that arise from totipotent cells; (3) embryos that arise from non-embryonic porcine cells; and (4) any of the above. The cloned porcine embryo is characterized by:

As used herein, the term “totipotent” as used with respect to embryos can refer to embryos that can develop into live birth pigs. The term "live birth" has been defined previously.

As defined herein, the term “cloned” refers to a cell having nuclear DNA substantially similar or identical to the nuclear DNA sequence of another cell, embryonic cell, fetal cell, and / or animal cell , Embryonic cells, fetal cells, and / or animal cells. The terms "substantially similar" and "matches" are described herein. A cloned embryo may result from a single nuclear transfer process, or alternatively, a cloned embryo may result from a cloning process that includes at least one recloning step. Since the recloning step can utilize embryonic cells isolated from embryos generated from totipotent cells, if the cloned embryo results from a cloning method that includes at least one recloning step, the cloned embryo is It can arise indirectly from totipotent cells.

In a preferred embodiment, (1) the cloned porcine embryo is a member of a number of embryos, wherein the number of embryos share substantially similar nuclear DNA sequences;
(2) The cloned porcine embryo is a member of a number of embryos, the number of embryos having the same nuclear DNA sequence; (3) the cloned porcine embryo is of a live birth pig Has a nuclear DNA sequence substantially similar to the nuclear DNA sequence; (4) one or more cells of the cloned porcine embryo have modified nuclear DNA; (5)
The cloned porcine embryo is subjected to an operation: (6) the operation includes culturing the embryo in a suitable medium; (7) the medium may contain feeder cells; Implanting the embryo into the reproductive tract of a female animal; (9) the female animal is preferably an ungulate, more preferably a porcine animal; (10) the estrous cycle of the female is Synchronized with the cycle; and (11) this operation involves incubating the embryo in an artificial environment.

All the preferred embodiments of the modified nuclear DNA for totipotent cells according to the invention extend to the cloned embryo according to the invention. In addition, any operations described in relation to the totipotent cells according to the invention apply to the cloned embryo according to the invention.

As used herein, the term “substantially similar” as used in reference to a nuclear DNA sequence refers to two approximately identical nuclear DNA sequences. The two sequences may differ due to differences in copy errors that usually occur during nuclear DNA replication. D substantially similar
NA sequences preferably match 97% or more, more preferably 98% or more,
And, most preferably, they match at least 99%. As used herein, the term "match" as used with respect to a nuclear DNA sequence can refer to the use of the phrase with respect to the amino acid sequence described herein above. Preferably, the nuclear DNA sequences are identical for the cloned animal and that is expected. Examples of methods for determining whether cloned animals and the cells from which they were cloned have substantially similar or matching nuclear DNA sequences are microsatellite analysis and DNA fingerprint analysis. Ashworth et al., 1998, Nature 394: 329 and Signer et al., 1998.
Nature 394: 329.

As used herein, the term “multiple” as used with respect to embryos refers to substantially similar nuclear DNA.
It can refer to a set of embryos having a sequence. In a preferred embodiment, the plurality is
5 or more embryos, 10 or more embryos, 15 or more embryos, 2
0 or more embryos, 50 or more embryos, 75 or more embryos, 1
It is composed of 00 or more embryos, and 1000 or more embryos.

As used herein, the term “culturing” as used with respect to embryos can refer to laboratory operations that include placing the embryos in a culture medium. The embryo can be placed in the culture medium for a suitable period of time to allow the embryo to quiesce or the embryo to grow in culture. Suitable culture media for culturing embryos are well known to those skilled in the art. For example, Nagashima et al., 1997, Mol
.Reprod. Dev. 48: 339-343; Petters and Wells, 1993, J. Reprod. Fert.
48: 61-73; Reed et al., 1992, Theriogenology 37: 95-109; Dobrinsky et al., 1996, Bio.
l.Reprod. 55: 1069-1074; U.S. Pat. No. 5,213,979, First et al., "In Vitro Culture of Bovine Embryos", May 25, 1993; U.S. Pat. No. 5,098,822, Rose.
See nkrans, Jr. et al., "Bovine Embryo Medium", March 17, 1992, each of which is hereby incorporated by reference in its entirety, including all drawings, tables, and drawings. Partial.

The term “suitable medium” as used herein can refer to any medium that allows for cell growth or embryo quiescence. If the medium causes cell growth, a suitable medium need not drive maximal growth, but only drive measurable cell growth. A suitable medium for embryo development may be an embryo culture medium as described by the examples herein. The term "feeder cells" has been previously defined herein. The embryo according to the present invention can be cultured in a medium with or without feeder cells. In another preferred embodiment, the feeder cells can be cumulus cells.

As used herein, the term “implantation” as used with respect to an embryo may mean inseminating a female animal with an embryo described herein. Implantation techniques are well known to those skilled in the art. See, for example, Polge and Day, 1982, “Embryo Transfer and Storage”, Control of
Pig Reproduction, DJA Cole and GR Foxcroft, London, UK, Butterwo
rths, pp. 227-291; Gordon, 1997, "Embryonic transfer and associated techniques in pigs", Con.
trolled reproduction in pigs (ed. by Gordon), CAB International, Wallingford, UK, pages 162-182; and Kojima, 1998, "Embryonic transfer", Manual of pig emb
ryo transfer procedures, National Livestock Breeding Center, Japanese So
See, ciety for Development of Swine Technology, pp. 76-79, each of which is incorporated herein by reference in its entirety, including all drawings, tables, and drawings.

Embryos can be developed in utero, or, alternatively, the fetus can be removed from the uterine environment before delivery.

As used herein, the term “tuned” as used with respect to the estrous cycle can refer to reproductive assistance techniques that are well known to those skilled in the art. These techniques are described in detail in the references cited in the preceding paragraph. Typically, estrogen and progesterone hormones are used to synchronize the estrous cycle of the female animal with the developmental stage of the embryo. As used herein, the term "developmental stage" can refer to embryos according to the present invention as well as morphological and biochemical changes during embryonic development. This developmental process is predictable for ungulate-derived embryos and can be synchronized with the estrous cycle of the recipient animal. Methods for synchronizing female pigs will be disclosed later.

The term “artificial environment” refers to an environment that promotes the development of an embryo or other growing cell mass. The artificial environment can be a uterine or fallopian tube environment of a species different from the species of the growing cell mass. For example, a developing bovine embryo can be placed in the uterus or fallopian tube of a sheep animal. Stice and Keefer, 1993, "Cloning of multi-generation bovine embryos", Biology of Reproduction 48: 715-719. Alternatively, the artificial development environment can be assembled in vitro. This type of artificial uterine environment can be synthesized using biological and chemical components known in the art.

In another aspect, the invention features a cloned mammalian embryo, wherein the embryo is totipotent, produced by a process comprising the step of nuclear transfer. Preferably, nuclear transfer occurs during (a) a nuclear donor, and (b) an oocyte, wherein the oocyte is at a stage capable of embryo formation.

In a preferred embodiment, (1) the oocyte is an enucleated oocyte; (2) the oocyte is preferably from an ungulate, more preferably from a porcine animal. (3) the oocyte is mature; (4) the oocyte is matured for more than 48 hours; (5) the oocyte is matured for about 53 hours; (6) the nucleus The donor is located in the perivitelline space of the oocyte; (7) The nuclear donor used for nuclear transfer is a cell described above (eg, a non-embryonic cell, a primordial germ cell, a germ cell, a differentiated cell, fetal cell Non-fetal cells, non-primordial germ cells, cells isolated from an asynchronous cell population, cells isolated from a synchronized cell population, cells isolated from an existing animal, embryonic stem cells, embryos Sexual germ cells, amniotic cells, cumulus cells, and fetal fibroblasts) (8) nuclear transfer can include the step of transposing a nuclear donor into a recipient oocyte; (9) transposing the nuclear donor into a recipient oocyte. (10) the transposition can include the step of fusion of a nuclear donor and an oocyte; (11) the fusion can include one or more fusions of the nuclear donor and the oocyte. Applying an electrical pulse can be included; (
12) fusion can include bringing the nuclear donor and the oocyte to at least one appropriate concentration of the fusion agent; (13) nuclear transfer includes the step of activating the nuclear donor and the oocyte (14) activation is achieved by (i) increasing intracellular levels of divalent cations in the cell and (ii) reducing phosphorylation of cellular proteins in the cell; (15) ) Activation is achieved by (i) introducing a divalent ionophore into the cell, and (ii) introducing a protein kinase inhibitor into the cell; (16) the divalent ionophore is a Ca ²⁺ ionophore; (17) the Ca ²⁺ ionophore is ionomycin; (18) the protein kinase inhibitor is DMAP; and (19) the activation
Achieved by introducing AP and ionomycin.

As used herein, the term “nuclear donor” can refer to a cell or a cell-derived nucleus that transposes into a nuclear acceptor. The nuclear donor may be a totipotent porcine cell. Further, the nuclear donor can be any cell described herein, including non-embryonic cells, non-fetal cells, differentiated cells, somatic cells, embryonic cells, fetal cells, embryonic stem cells, primordial germ cells Germ ridge cells, cumulus cells, amniotic cells, fetal fibroblasts, hepatocytes, embryonic germ cells, adult cells, cells isolated from asynchronous cell populations, and cells isolated from synchronized cell populations [Where the synchronized population is not arrested in the _G0 phase of the cell cycle], but is not limited thereto. The nuclear donor cells further
It may be a cell differentiated from an embryonic stem cell. For example, Piedrahita et al., 1998, Biol
Reprod. 58: 1321-1329; Shim et al., 1997, Biol. Reprod. 57: 1089-1095; Tsung et al., 1995, Shih Yen Sheng Wu Hsueh Pao 28: 173-189; and Wheeler, 1994, Rep.
6: 563-568, each of which is hereby incorporated by reference in its entirety, including all drawings, drawings, and tables. in addition,
The nuclear donor may be a previously frozen or cryopreserved cell.

[0114] As used herein, the term "enucleated oocyte" can refer to an oocyte whose nucleus has been removed. Typically, the needle can be placed in the oocyte and the nucleus aspirated into the needle. This needle can be withdrawn from the oocyte without breaking the cytoplasmic membrane. This enucleation technique is well known to those skilled in the art. See U.S. Patent No. 4,994,384; U.S. Patent No. 5,074,420; and Willadsen, 1986, Nature 320: 63-65. The enucleated oocytes are preferably mature for more than 24 hours, preferably 3 hours.
It is prepared from oocytes that have matured for 6 hours or more, more preferably for 48 hours or more, and most preferably for about 53 hours.

As used herein, the terms “mature” and “mature” can refer to the process of incubating an oocyte in culture in vitro. The maturation medium is
Many types of components can be included, including hormones and growth factors. The time of maturation is determined from the time the oocyte is placed in the maturation medium to the time the oocyte is subjected to manipulation (eg, enucleation, nuclear transfer, fusion, and / or activation). be able to. Oocytes can be matured in many media well known to those skilled in the art. See, for example, Mattioli et al., 1989, Theriogenology 31: 1201-1207; Jolli.
ff and Prather, 1997, Biol. Reprod. 56: 544-548; Funahashi and Day, 1993.
98: 179-185; Nagashima et al., 1997, Mol. Reprod. Dev. 38: 339-3.
43; Abeydeera et al., 1998, Biol. Reprod. 58: 213-218; Funahashi et al., 1997, Biol.
57: 49-53; and Sawai et al., 1997, Biol. Reprod. 57: 1-6, each of which is incorporated by reference in its entirety, including all drawings, tables, and drawings. Part of this specification. The oocyte can be matured for any time: the oocyte can be matured for at least 10 hours, at least 20 hours, at least 24 hours, at least 60 hours, at least 72 hours, at least 90 hours, preferably at least 36 hours More preferably, more than 48 hours, and most preferably about 53 hours.
The term "about" with respect to oocyte maturation can refer to plus or minus 3 hours.

[0116] Oocytes can also be matured in vivo. The time of maturation can be from the time the oocyte receives the appropriate stimulus to resume meiosis to the time the oocyte undergoes manipulation. Maturation times similar to those described above for oocytes to be matured in vitro apply to oocytes to be matured in vivo.

[0117] A variety of oocytes can be selected for maturation. For example, an oocyte
It can be isolated from pre-adolescent pigs or near adolescents (eg, sows). However, oocytes from pre-adolescent pigs may not be able to resume spontaneous meiosis in vitro. It is a preferred embodiment of the present invention that the oocytes isolated from the sow are used for maturation and finally used for nuclear transfer. Nuclear transfer can be achieved by combining one nuclear donor and one or more enucleated oocytes. In addition, nuclear transfer can be achieved by combining the cytoplasm of one nuclear donor, one or more enucleated oocytes, and one or more enucleated oocytes.

[0118] As used herein, the term "cybrids" can refer to an oocyte into which a nuclear donor has been inserted. The term "cybrid" can refer to an oocyte having a nuclear donor transposed into an oocyte. A nuclear donor can be fused with an oocyte, and the term "cybrids" includes oocytes that have not been fused with a nuclear donor.

The present invention relates, in part, to cloned mammalian embryos established by nuclear transfer of a nuclear donor and a non-enucleated oocyte. A cloned embryo can be established if the nuclear DNA from the oocyte does not replicate and the nuclear DNA from the donor cell replicates during cell division. See, for example, Wagoner et al., 1996, "Functional enucleation of bovine oocytes; effects of centrifugation and ultraviolet light", Theriogenology 46: 279-284.

The term “another ungulate” as used herein refers to a situation where the nuclear donor has an origin in an ungulate of a species, tribe or family different from that of the recipient oocyte. be able to. For example, porcine cells can be used as a nuclear donor, while recipient oocytes can be isolated from cattle. This example is not meant to be limiting,
Combinations of ungulates of any species / family of nuclear donor and recipient oocytes are contemplated by the present invention.

As used herein, the term “transposition” as used in reference to nuclear transfer can refer to combining a nuclear donor and a recipient oocyte. Transposition can be performed by techniques such as fusion and / or direct injection.

As used herein, the term “injection” as used with respect to an embryo may mean piercing the oocyte with a needle and inserting the nuclear donor in the needle into the oocyte. In a preferred embodiment, the nuclear donor can be injected into the cytoplasm of the oocyte or into the perivitelline space of the oocyte. This direct injection approach is well known to those skilled in the art, as pointed out by the publications already incorporated herein for nuclear translocation. For a direct injection approach toward nuclear transfer, whole cells can be injected inside the oocyte, or, alternatively, nuclei isolated from the cell can be injected inside the oocyte. Such an isolated nucleus may be surrounded only by the nuclear envelope, or the isolated nucleus may be surrounded by the nuclear envelope and the plasma membrane in any proportion. Oocytes can be pretreated to increase the strength of their plasma membrane, for example, by incubating the oocytes in sucrose prior to injection of a nuclear donor.

Techniques for placing a nuclear donor into the perivitelline space of enucleated oocytes are well known to those of skill in the art, and are described in detail herein in the patents and references cited above for nuclear transfer. ing.

As used herein, the term “fusion” may mean joining portions of a lipid membrane corresponding to a nuclear donor and a recipient oocyte. The lipid membrane corresponds, for example, to the plasma or nuclear membrane of the cell. Fusion occurs when, for example, the nuclear donor and the recipient oocyte are positioned adjacent to each other, or when the nuclear donor is positioned in the perivitelline space of the recipient oocyte, with the addition of a fusion stimulus. Can happen.
Specific examples for translocating mammalian pig cells inside oocytes are described below in other preferred embodiments. These techniques for transposition are described in detail herein in the references cited above for nuclear translocation.

As used herein, the term “electrical pulse” may refer to passing an electrical current through a nuclear donor and a recipient oocyte. For nuclear transfer, the nuclear donor and recipient oocytes are aligned between electrodes and a current is applied. The current can be alternating or direct current. The current is delivered to the cell as a single pulse or as multiple pulses at various different times. Cells are typically cultured in a suitable medium for delivery of electrical pulses. Examples of electrical pulse conditions utilized for nuclear transposition are described herein in the references and patents cited above for nuclear transposition.

As used herein, the term “fusion agent” refers to the probability that a portion of the plasma membrane from a different cell will fuse when a nuclear donor is located adjacent to a recipient oocyte. Can refer to any compound or organism that can increase the In a preferred embodiment, the fusogenic agent is polyethylene glycol (PEG), trypsin, dimethylsulfoxide (DMSO), lectin, agglutinin, virus,
And Sendai virus. These examples are not intended to be limiting and other fusogenic agents known in the art are applicable and encompassed by the present invention.

As used herein, the term “appropriate concentration” as used in reference to a fusion agent can refer to the concentration of the fusion agent that provides a measurable amount of fusion. Fusion can be measured by a number of techniques well known to those skilled in the art, for example, utilizing light microscopy, dyes, and fluorescent lipids.

The term “activation” can refer to materials and methods useful for stimulating and dividing cells before, during, and after the nuclear transfer step. The term "cell" used in the previous sentence can refer to oocytes, cybrids, nuclear donors, and early embryos. These types of cells may require stimulation to divide after nuclear transfer has occurred. The present invention relates to any activating materials and methods known to those skilled in the art.

The electrical pulse is sometimes sufficient to stimulate activation of the cell, but for proper activation of the cell, other non-electrical means of activation are useful, and Often needed. Useful chemical materials and methods for non-electrical activation are described below in other preferred embodiments of the invention. When two or more chemical components are introduced into a cell for activation of the cell, the components can be added simultaneously or stepwise.

[0130] Examples of electrical processes for activation are well known in the art. Researchers have also reported non-electrical processes for activation. For example, Grocholova et al., 19
97, J. Exp. Zoology 277: 49-56; Schoenbeck et al., 1993, Theriogenology 40: 257-2.
66; Prather et al., 1989; Biology of Reproduction 41: 414-418; Prather et al., 1991.
, Molecular Reproduction and Development 28: 405-409; Mattioli et al., 1991, M.
olecular Reproduction and Development 30: 109-125; Terlouw et al., 1992, Theri
ogenology 37: 309; Prochazka et al., 1992, J. Reprod. Fert. 96: 725-734; Funahash
i et al., 1993, Molecular Reproduction and Development 36: 361-367; Prather et al., Bio.Rep.
Development 41: 70-75; Funahashi et al., 1995, Zygote 3: 273-281; Wang et al., 1997.
, Biology of Reproduction 56: 1376-1382; Piedrahita et al., 1989, Biology of R.
eproduction 58: 1321-1329; Machaty et al., 1997, Biology of Reproduction 57:85.
-91; and Machaty et al., 1995, Biology of Reproduction 52: 753-758.

Examples of components useful for non-electrical activation include ethanol; inositol triphosphate (IP ₃ ); Divalent ions (eg, Ca²⁺And / or Sr²⁺Addition); microtubules
Inhibitors (eg, cytochalasin B); ionophores for divalent ions
(For example, Ca²⁺Ionophore ionomycin); protein kinase inhibitor (
For example, 6-dimethylaminopurine (DMAP)); a protein synthesis inhibitor (eg,
For example, cyclohexamide); phorbol 12-myristate 13-acetate (P
Phorbol esters such as MA): and sapshigargin. Temperature change
And mechanical techniques are also known to be useful for non-electrical activation.
You. The present invention includes any activation technique known in the art. For example, "Parthagen
U.S. Patent No. 5,496,720 to Susko-Parrish et al. Entitled "Oocyte Activation"
(Registered March 5, 1996), and Wakayama et al., 1998, Nature 394: 369-374.
Each of these, by reference, includes by reference all drawings, tables, and drawings
The entire contents of the description are incorporated herein by reference.

When utilizing ionomycin and DMAP for non-electrical activation, ionomycin and DMAP can be introduced into the cells simultaneously or by stepwise addition, the latter being used in a preferred manner as described herein. is there. Preferred concentrations of ionomycin and DMAP are from 0.5 μM ionomycin to 50 μM ionomycin, and from 0.5 mM DMAP to 50 mM DMAP, more preferably from 1 μM ionomycin to 20 μM ionomycin, and 1 mM DMAP.
5 mM DMAP, and most preferably about 10 μM ionomycin and about 2 mM DMAP [wherein the term “about” can refer to plus or minus 2 μM ionomycin and 1 mM DMAP].

In another preferred embodiment, (1) one or more cells of the cloned porcine embryo contains modified nuclear DNA; (2) manipulating the cloned porcine embryo; A) separating the at least one individual cell from the cloned embryo; (4) the step including utilizing the individual cell as a nuclear donor in a nuclear transfer method; (6) separating the individual cells from the preblastocyst stage embryo; and (7) separating the individual cells from the preblastocyst stage embryo. (8) the recloning process comprises: (a) separating the embryo into one or more individual cells, and (b) (i) individual cells of (a), and (ii) oocytes. Nuclear transfer during (9) placing individual cells into the perivitelline space of enucleated oocytes for subsequent nuclear transfer; (10) subsequent nuclear transfer Comprising at least one of the steps of transposition, injection, fusion, and activation of the cells and / or enucleated oocytes; (11) cloned mammalian embryo cells resulting from this subsequent nuclear transfer One or more of which comprises the modified nuclear DNA; and (12) the cloned mammalian embryo resulting from this subsequent nuclear transfer can be subjected to subsequent manipulations, wherein Subsequent manipulation is any of the manipulation steps previously defined herein for totipotent cells and / or cloned embryos].

The term “individual cell” as used herein can refer to a cell isolated from a cloned mammalian embryo according to the present invention. Individual single cells can be isolated from embryos by techniques well known to those skilled in the art, as described in the references cited herein above.

As used herein, the term “subsequent nuclear transfer” is also referred to as a “recloning” step. Preferably, a recloning step can be utilized to enhance nuclear reprogramming during nuclear transfer such that the product of nuclear transfer is a live birth animal. The number of subsequent nuclear transfer steps will be described in more detail later.

[0136] Any of the preferred embodiments for the transposition, injection, fusion, and activation steps described herein above may relate to any of the subsequent nuclear transfer steps.

The term “inner cell mass” as used herein can refer to cells that give rise to the embryo itself. The cells that line the outside of the blastocyst can be referred to as the trophoblast of the embryo.
Methods for isolating the inner cell mass from the embryo are well known to those skilled in the art, as described above. "
The term "problastocyst" is well known to those skilled in the art and has been mentioned above.

As used herein, the term “ovulated in vivo” refers to a number of hours after the animal has exhibited estrous properties or after injection of an exogenous gonadotropin known to induce ovulation. Oocytes isolated from the animal.
The nature of animals in estrus is well known to those skilled in the art, as described in the references disclosed herein. See, eg, Gordon, 1977, “Embryonic transfer and associated techniques in pigs (Gordon)”, CAB International, Wallingford, UK, pp. 60-76.
And Kojima, 1988, Embryo transfer, Manual of pig embryo transfer procedures
, National Livestock Breeding Center, Japanese Society for Development o
f See Swine Technology, pp. 7-21, each of which is incorporated herein by reference in its entirety, including all drawings, tables, and drawings.

In another embodiment, the present invention provides a method comprising: (a) transposing a nuclear donor into an oocyte to establish a nuclear transfer oocyte; and (b) a nucleus to establish a porcine embryo. The present invention relates to a cloned porcine embryo produced by a process comprising the step of non-electrical activation of metastatic oocytes.

In a preferred embodiment, (1) the nuclear donor is a cultured cell and is selected from any of the cell types described herein; (2) the nuclear donor is a totipotent cell or a totipotent cell (3) the nuclear donor is any of the cell types described herein (eg, embryonic germ cells, cumulus cells, amniotic cells, fibroblasts); (4) transposition is a fusion step And (5) the process comprises culturing the embryo in vitro. Any of the other preferred embodiments described herein with respect to porcine embryos, and particularly with regard to activation, relate to aspects of the present invention.

[0141] In another aspect, the invention relates to a method of producing a cloned porcine embryo. The method comprises: (a) a nuclear donor, wherein the nuclear donor is a totipotent porcine cell;
And (b) a step of nuclear transfer during the oocyte, wherein the oocyte is at a stage where embryo formation is possible. In yet another aspect, the invention provides a nuclear transfer egg comprising: (a) transposition of a nuclear donor into an oocyte to establish a nuclear transfer oocyte; and (b) a nuclear transfer egg to establish a porcine embryo. The present invention relates to a method for cloning a porcine embryo, which comprises a step of non-electrical activation of a mother cell. In a preferred embodiment, any aspect of the invention relating to a cloned porcine embryo applies to a method for producing a cloned porcine embryo.

In another aspect, the invention features a cloned porcine embryo derived from a totipotent embryo according to the invention. The fetus can be isolated from the uterus of a pregnant female animal and, in the case of an ectopic pregnancy, from other parts of the pregnant female animal.

[0143] In a preferred embodiment, (1) one or more cells of the fetus have altered nuclear DNA (as defined herein above); and (2) the fetus is as defined herein. It can be attached to any operation performed. For example, one operation involves isolating a fetus from the uterus of a pregnant female animal, isolating cells from the fetus (eg, primordial germ cells), and using the isolated cells as a nuclear donor for nuclear transfer , A step.

Another aspect of the present invention relates to (1) (a) production of a cloned porcine embryo according to the above definition, and (b) a cloned porcine embryo such that the cloned porcine embryo develops into a fetus. Manufactured by a process that includes the steps of
(2) (a) producing a cloned porcine embryo as defined above, and (b) manipulating the cloned porcine embryo so that the cloned porcine embryo develops into a fetus. A method for producing a cloned porcine fetus, comprising the steps of: (3) isolating at least one cell type from the fetus; And (4) using the cloned fetus of the present invention (eg, for establishing a feeder cell layer), comprising the step of separating at least a portion of the fetus into individual cells. And

Cloned Pigs According to the Invention In another aspect, the invention features cloned pigs derived from totipotent pig cells according to the invention. Cloned pigs can be developed from cloned embryos established by the process of nuclear transfer between totipotent porcine cells and oocytes. Totipotent porcine cells are preferably established by utilizing any of the materials and methods described herein above.

[0146] In yet another embodiment, the invention relates to a cloned swine animal, wherein the animal is a member of a number of swine animals, and the plurality of animals are substantially similar in nuclear DNA. Has an array. The term "substantially similar" in relation to a nuclear DNA sequence has been defined previously herein.

[0147] In a preferred embodiment, (1) the majority comprises 5 or more animals, 10 or more animals, 100 or more animals, and 1000 or more animals; and (2) The multiple animals can have the same nuclear DNA sequence. The term "identical" with respect to a nuclear DNA sequence has been described herein above.

In another aspect, the invention is directed to a cloned porcine animal having one or more cells containing the modified nuclear DNA. All of the preferred embodiments described above for the modified nuclear DNA apply to the cloned porcine animals of the present invention.

In yet another aspect, the invention features a method of using a cloned porcine animal, comprising the step of isolating at least one component from the porcine animal.

The term “component” as used herein may relate to any part of a pig animal. The components can be selected from the group consisting of fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses. For example, progenitor cells as defined above can be derived from fluids, biological fluids, cells, tissues, organs, gametes, embryos, and fetuses isolated from the cloned organisms of the present invention.

As used herein, the term “gamete” can refer to any cell that participates directly or indirectly in an animal's reproductive system. Gametes can be specialized products from the gonads of the living body, where the gametes can transfer genetic material while participating in insemination. Examples of gametes are spermatocytes, spermatogonia, oocytes, and oocytes. Gametes can be present in fluids, tissues, and organs collected from animals (eg, sperm are present in semen). The present invention relates to the collection of any type of gametes from animals. For example, methods for collecting semen and oocytes are known to those skilled in the art. For example, Gordon, 1997, "Introduction to Pig Breeding Management, Pig Embryo Transfer and Associated Technologies", Controlled reproduction in pigs (Gordon), CAB International,
Wallingford, UK, 1-59; Mattioli et al., 1989, Theriogenology 31: 1207.
98 179-185; Funahashi et al., 1997, Biol. Reprod. 57: 49-53; Abeydeera et al., 1998, Biol. Reprod. 58: 213-21.
8; and Sawai et al., 1997, Biol. Reprod. 57: 1-6, each of which is hereby incorporated by reference in its entirety, including all drawings, tables, and drawings. And

The term “organization” has been defined above. The term "organ" refers to any organ or any part of an organ isolated from an animal. Examples of organs and tissues are nerve tissue, brain tissue, spleen, heart, lung, gall bladder, pancreas, testis, ovary and kidney. These examples are not limiting and the present invention relates to any organ and any tissue isolated from the cloned animal according to the present invention.

In a preferred embodiment, the invention relates to (1) fluids, biological fluids, cells, tissues,
Subjecting the organs, gametes, embryos, and fetuses to an operation; (2) the operation comprises:
And / or may include cryopreserving the fetal tissue; (3) the operation may include thawing the cryopreserved; (4) the operation may involve semen removal from the X chromosome. (5) the operation includes a method of preparing semen for artificial insemination; (6)
This operation includes the step of purifying the desired polypeptide from the biological fluid or tissue; (7) This operation involves the concentration of the biological fluid or tissue; The method can further include transferring the further fluid, cloned cells, cloned tissue, cloned organ, and / or a portion of the cloned organ to a recipient organism (eg, the recipient organism (9) the recipient organism is non-human; and (10) the recipient organism is a human.

As used herein, the term “separating” as used with respect to semen separation can refer to methods well known to those skilled in the art for fractionating a semen sample into sex-specific fractions. This type of separation can be accomplished using a commercially available flow cytometer.
The use of flow cytometers to separate sperm by gene content is well known in the art. In addition, semen can be separated by paragenesis by other methods well known to those skilled in the art. US Pat.
39362, 5346990, and 502,244 (registered August 8, 1995, September 13, 1994, and June 4, 1991, respectively), each of which is incorporated by reference into all drawings, tables, And the entire contents including the drawing are part of this specification.

[0155] As used herein, the term "purification" can mean increasing the specific activity of a particular polypeptide or group of polypeptides in a sample. Specific activity can be expressed as the ratio between the activity or amount of the target polypeptide and the concentration of the total polypeptide in the sample. The activity may be, for example, catalytic activity and / or binding activity. Specific activity can also be expressed as a ratio between the concentration of the target polypeptide and the concentration of the total polypeptide. Purification methods include dialysis, centrifugation, and column chromatography techniques, which are methods well known to those skilled in the art. See, e.g., Young et al., 1997, "Biopharmaceutical protein production in milk of transgenic dairy animals", BioPharm 10 (6): 34-38.

[0156] As used herein, the term "metastatic" may relate to the transfer of a group of cells, tissues, organs, and / or parts of an organ to an animal. The cells, tissues, organs, and / or portions of the organs can be, for example, (a) grown in vitro and then transferred to an animal, and (b) excised from a cloned porcine animal and treated with another species of a different species. Transferred to an animal, (c) excised from a cloned pig animal, transferred to another animal of the same species, and (d) excised from a portion of the animal (eg, cells from an animal's leg). Can then be transferred to another part of the same animal (eg, the brain of the same animal) and / or (e) any combination of the above.

As used herein, the term “translocating” refers to adding a fluid, cell, tissue, and / or organ to an animal, and removing cells, tissue, and / or organ from the animal. And may mean exchanging for cells, tissues, and / or organs from another source. For example, to treat a neurological disease (eg, Alzheimer's disease), the cloned porcine tissue-derived neural tissue can be transplanted into an appropriate region of the human nervous system. In another example, a heart or a portion of a heart can be removed from a cloned porcine animal and surgically inserted into a human who has previously had the heart or a portion of the heart removed. Surgical methods for achieving this preferred embodiment of the present invention are known to those of ordinary skill in the art of surgery. The transfer method can include the step of removing cells, tissues, fluids and / or organs from the recipient organism prior to the transfer step.

In another embodiment, the invention relates to (1) (a) producing a cloned porcine embryo by any one of the methods described herein for producing a cloned porcine embryo; And (b) a cloned porcine animal produced by a process comprising the step of manipulating the cloned porcine embryo such that the cloned porcine embryo develops into a live birth animal; and (2) (A) according to any one of the methods described herein for producing a cloned porcine embryo,
Producing a cloned porcine embryo, and (b) manipulating the cloned mammalian embryo such that the cloned mammalian embryo develops into a live birth pig animal. A process for cloning an animal.

In a preferred embodiment, (1) the operation can include the step of implanting the embryo into the uterus of the animal; (2) the estrous cycle of the animal can be synchronized with the developmental stage of the embryo. And (3) the operation can include the step of implanting the embryo in an artificial environment.

In another embodiment, the invention provides (a) translocating a nuclear donor into an oocyte to establish a nuclear transfer oocyte; (b) making the nuclear transfer oocyte non-electrically active Establishing a cloned porcine embryo; and (c) transferring the porcine embryo into a recipient female, where the porcine embryo develops into a cloned porcine animal,
Characterized by a process for cloning a pig animal.

[0161] In a preferred embodiment, (1) the nuclear donor is a cultured cell and is selected from any of the cell types described herein; (2) the nuclear donor is a totipotent cell;
) Transposition comprises fusion; and (4) the method comprises culturing the porcine embryo in vitro. Any other preferred embodiments described herein with respect to porcine embryos, and particularly with regard to activation, are all relevant to this aspect of the invention.

The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, and from the claims. .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates aspects of the invention relating to generating totipotent cells from progenitor cells. This figure shows that totipotent cells can be generated from embryonic stem cells, primordial germ cells, and cells isolated from animals.
The source of progenitor cells shown in FIG. 1 is not limiting, and other sources of progenitor cells are described herein. FIG. 2 illustrates aspects of the invention with respect to totipotent cell cell lines and routes for establishing clonal animals. Fibroblasts can be isolated from any of the sources described herein. FIG. 3 illustrates aspects of the invention for establishing a cloned transgenic cell line and a cloned transgenic animal.

(Detailed Description of Preferred Embodiments) The present invention relates to a cloning technique for pig animals. The present invention provides a variety of advantages over the means and methods currently used in the field of pig cloning. For example, the present invention relates in part to totipotent cells useful for cloning pig animals. These totipotent cells can be the source of methods for producing porcine cloned animals by utilizing virtually any type of cell. For example, cells isolated from live born animals can be reprogrammed into totipotent cells. This feature of the invention allows one to easily assess the phenotype of an existing porcine animal and then establish a totipotent cell line for cloning the animal.

In addition, the totipotent cells of the present invention allow cell lines to be established from virtually any type of cell. This reprogramming method has been described earlier in this specification. These totipotent cell lines provide an almost unlimited source of donor cells for nuclear transfer cloning techniques. In addition, because of the lower rate of differentiation of these cell lines than existing cell lines used for cloning, this feature
The advantage is provided that the cloning efficiency is increased. For example, an embryonic stem cell line may contain a large number of colonies of cells that are not totipotent. The cell lines of the invention may contain a high percentage of totipotent cells.

In addition, cloning efficiency is increased by the methods and processes for establishing totipotent cells, totipotent cloned embryos and cloned animals of the present invention. This increased efficiency meets the long-awaited needs in the art.

I. Totipotent porcine cells A. Establishing Totipotent Cells The totipotent cells of the present invention can be produced from any type of progenitor cells. A preferred embodiment of the present invention relates to the following types of progenitor cells. That is,
(1) embryos resulting from two gametes that have merged in vitro or in vivo, (2)
Embryonic stem cells (ES cells) arising from cultured embryonic cells (eg, pre-undifferentiated germ cells and inner cell mass cells), (3) cultured and uncultured inner cell mass cells isolated from embryos,
(4) cultured and non-cultured undifferentiated germ cells, (5) cultured and non-cultured fetal cells,
(6) cultured and uncultured primordial germ cells, (7) cultured embryonic germ cells (EG cells) as defined herein, (8) cultured and uncultured cells isolated from animals,
9) cultured and non-cultured cumulus cells, (10) cultured and non-cultured amniotic cells, (11) cultured and non-cultured fetal fibroblasts, (12) cultured and non-cultured germ protuberance cells, (1)
3) Cultured and non-cultured differentiated cells, and (14) Cultured and non-cultured undifferentiated cells or undifferentiated cells.

The totipotent cells of the invention are preferably generated from the progenitor cells specifically described herein. Cells from the pig animal can be isolated from almost any type of tissue. For example, ear perforation specimens can be taken from a pig animal, cells dissociated from the specimen, and the cells subsequently cultured in vitro by using cell culture techniques familiar to those of ordinary skill in the art. Examples of materials and methods for culturing primary cultured cells into totipotent cells are described in the embodiments below.

A variety of cell culture methods exist in the art. For example, Culture of animal cell
s: a manual of basic technique (second edition), Freshney, copyright 1987 (Alan
R. Liss, Inc., New York). In particular, cells that are precursor cells of totipotent cells, and totipotent cells themselves, can be cultured in a feeder layer. Examples of feeder layers are well known to those of ordinary skill in the art and can be generated from a number of different cells cultured in vitro. For example, the embodiment described below and Strelch
enko, 1996, Theriogenology 45: 130-141; Piedrahita et al., 19
90, Theriogenology 34: 879-901; Piedrahita et al., 1998, Biol.
Reprod. 58: 1321-1329; and Shim et al., 1997, Theriogenolog.
y 47: 245 (each incorporated herein by reference, including schematics, tables and drawings). However, the precursor cells of the totipotent cells and the totipotent cells themselves need not be cultured in the feeder cell layer.

Preferred culture conditions for these progenitor cells are cell culture media containing significant amounts of glucose, in the amounts specifically set forth herein. Cell culture conditions can include carbohydrates that are different from glucose and can include many types of carbohydrates and complex carbohydrates. A wide variety of carbohydrates are well known to those of ordinary skill in the art. See, for example, Sigma and DIFCO catalogs.

Preferred cell culture conditions also relate to a cell culture medium containing one or more antibiotics. Antibiotics suitable for use in cell culture media are known in the art. For example, Culture of Animal Cells: a manual of basic technique
s (3rd edition), 1994, Freshney (ed.), Willie-Lith, Inc .; Cells: a laboratory manual (1st volume), 1998, Spector, Goldman.
, Leinwand (ed.), Cold Spring Harbor Laboratory Press, and Animal Cells: culture and media, 1994, Darling & Morgan, John
See Willie and Sons, Limited, each of which is hereby incorporated by reference, including diagrams, tables and drawings.

Another example of cell culture conditions is a cell culture medium containing one or more recipient ligands. Examples of recipient ligands are familiar to the average technician in the art. Cytokines and / or growth factors are preferred recipient ligands of the present invention. See, for example, the R & D Systems Catalog (614 McKinley Place NE, Minneapolis, Minn. 55413). In embodiments, progenitor cells can be reprogrammed into totipotent cells by adding varying amounts of human recombinant leukemia inhibitory factor (hrLIF) and basic bovine fibroblast growth factor (bFGF) to the culture medium. Various concentrations of these two cytokines are preferably at a concentration of 1-1000 ng / mL, more preferably 10-500 ng / mL.
/ ML, and most preferably at a concentration of about 100 ng / mL to the culture medium. Exogenous soluble and membrane-bound forms of the steel factor are not required to convert progenitor cells into totipotent cells.

These examples are not meant to be limiting, and cytokines or combinations of cytokines may be added or deleted from those described in the embodiments below. Preferred cytokines for generating totipotent cells include fibroblast growth factor (FGF), leukemia inhibitory factor (LIF), cardiotrophin (cardiotrophin).
) 1 (CT-1), ciliary neurotrophic factor (CNTF), stem cell factor (SCF),
It may be selected from the group consisting of oncostatin M (OSM) and members of the interleukin (IL) group, including IL-6, IL-11 and IL-12.

By adding or removing other cytokines and other molecules other than cytokines from the recipient ligand cocktail described in the embodiments below,
Totipotent cells can be established from any of the cells described in the preceding section. Modifications may be made to any of the totipotent cell generation conditions from those described herein. The ability of these modified totipotent cell generation conditions can be monitored by the methods specified in the section "Identifying Totipotent Cells" below.

B. Identification of Totipotent Cells Totipotent cells can be identified in several ways. Examples of tests for cell totipotency include:
(1) identification of markers specific to totipotent cells; (2) performance of one or more nuclear transfer cycles by the cells (described below) and development of the generated embryos into animals.

By utilizing markers, non-totipotent cells and totipotent cells can be distinguished.
Markers can be selected from the group consisting of low molecular weight markers, macromolecular markers, cell surface markers and intracellular markers. Examples of markers that may be suitable for identifying totipotent cells may be selected from the group consisting of alkaline phosphatase, cytokeratin, vimentin, laminin and c-kit. These markers are familiar to the average person skilled in the art and these examples are not intended to be limiting.

[0177] Some of these markers have been tested on cultured bovine cells that have been identified for totipotency. As described above, the totipotent porcine cells of the present invention cannot appreciably stain for alkaline phosphatase. Thus, the cells of the invention contrast with the pluripotent cells discussed in the publications cited above. Since some of the markers listed above may be present in both pluripotent and totipotent cells, some of these markers may not be specific for totipotent cells It should be noted.

The present invention relates to any markers specific for totipotent cells known to the average person skilled in the art. Markers for totipotency that are not clearly defined in the art can be elucidated by processes such as differential expansion and genomic methods for elucidating totipotent cell markers. Totipotent cells also
Cells can be identified by subjecting them to nucleic acid sequence content analysis (eg, hybridization techniques with nucleic acid probes). Nucleic acid samples from totipotent cells and nucleic acid samples from non-totipotent cells can be screened for specific nucleic acid sequences. Where a sample from a non-totipotent cell lacks a nucleic acid sequence present in the totipotent cell, the nucleic acid sequence may be a marker for distinguishing the totipotent cell from the non-totipotent cell. Similarly,
If the specimen from the non-totipotent cell contains a nucleic acid sequence that is not present in the totipotent cell, the nucleic acid sequence can be a marker for distinguishing the totipotent cell from the non-totipotent cell. By utilizing polypeptide analysis techniques (eg, PAGE, SDS-PAGE, methods involving antibodies, and HPLC techniques known in the art), polypeptide markers can be elucidated in a similar manner.

A preferred test for cellular totipotency measures whether the cells give rise to totipotent embryos and, in effect, clonal animals. This test represents a typical test for cell totipotency. One example of such a test includes the following steps. That is, (1) utilizing latent totipotent cells for nuclear transfer with enucleated oocytes, (2) developing the generated cybrids, and (3) separating embryos that have developed from the cybrids into individual cells. Subjecting one or more of the individual cells to a second round of nuclear transfer, (
4) developing the cybrid generated from step (3) into an embryo; (5) step (2)
Or implanting the embryo from (4) into the uterine environment and (6) developing the embryo. If the resulting fetus grows past the first trimester of gestation, the cells initially used for nuclear transfer are likely to be totipotent cells. The cells are clearly totipotent if the cells used for nuclear transfer grow into live, cloned animals. Examples of techniques (eg, oocyte enucleation and nuclear transfer) utilized in this exemplary test are well described in the literature and in the embodiments described below.

Using the tests to identify totipotent cells, the materials and (methods) described herein can be modified by the average technician in the art to obtain totipotent cells from any type of progenitor cells. Cells can be generated. Thus, the present invention is intended to allow any average technician in the art to easily produce totipotent cells by utilizing the materials and methods described herein in conjunction with totipotent cell identification methods. Includes all of the materials and methods described herein and modifications to these totipotent cell generation methods.

C. Identification of Totipotent Cells That Are Permanent Cells The materials and methods described above (eg, culturing cells with cytokines) can convert non-permanent cells to permanent cells. There are other methods in the art for generating permanent cell lines from primary cells and for identifying permanent cells. For example, cells can be immortalized by manipulating telomerase activity within the cell. For example,
West et al., July 8, 1997, "Therapy and Diagnosis of Conditions Rela.
US Patent No. 5, entitled "ted to Telomere Length and / or Telomerase Activity"
See US Pat. No. 6,459,986, including all schematics, drawings and tables, which are hereby incorporated by reference.

[0182] Permanent cells can be identified by measuring the number of times cell division occurs in cultured cells and the number of cells doubles before cell termination. As discussed above, permanent cells may have doubled 10, 20, 30, 40, 50, and 60 times before cell termination. Materials and methods for measuring cell termination have been set forth above.

Further, permanent cells can be identified by detecting the presence and / or absence of low molecular weight and macromolecular markers that are specific for permanent cells. The presence or absence of the marker may be a determinant of cell immortalization. Further, the phenomena associated with the marker may be indicative of immortality. For example, where the marker is an enzyme, an indication of the presence of the enzyme and / or some level of catalytic activity of the enzyme may be a suitable indicator that certain cells are permanent.

[0183] Low molecular weight markers include specific nucleosides, lipid-bound sialic acids, polyamines, and pseudouridine. These examples are not limiting and the invention encompasses any other low molecular weight markers known in the art.

[0185] Macromolecular markers include nucleic acid polymers, peptides, polypeptides, proteins,
It can be divided into several classes, including enzymes, growth factors, growth factor recipients, hormones, hormone recipients, oncogenes, oncogene products and specific glycoproteins. The macromolecular marker may be selected from the group consisting of an extracellular protein, a membrane bound protein and / or an intracellular protein, which may be membrane bound or soluble. One such marker for permanent cells is, for example, telomerase or its concomitant activity. See U.S. Patent No. 5,645,986 (supra). Other examples of permanent cell specific markers can be selected from the list below. These examples are not limiting and the invention is intended to include all permanent cell specific markers known in the art.

1) Epidermal growth factor (EGF) and its recipient (EGF-R) 2) Transforming growth factor-alpha (TGF-alpha) and its recipient 3) c-erbB2 recipient tyrosine kinase (HER2 product) 4) Hyaluronan recipient (possibly CD44, integral membrane glycoprotein) 5) Carcinoembryonic antigen (CEA) group of tumor markers (eg T1, glycosylated protein) 6) Telomerase, maintaining telomere length in permanent cells Ribonucleoprotein 7) Phosphatases: placental alkaline phosphatase (PLAP), germline alkaline phosphatase, prostate acid phosphatase (PAS) 8) Cathepsin D (catalyzes the degradation of laminin) 9) Ornithine decarboxylase (ODC) (POC) Catalyzes the rate-limiting step in the amine synthesis) 10) Beta - glucuronidase 11) Alpha -6 integrin 12) Keratin K8 13) oncogene products: ras oncogene (k-ras, Ha-ras, p21)
, -Src, c-myc 14) Cyclin D1, cyclin A, and retinoblastoma gene protein (R
b) 15) Changes in p53 expression or p53 mutations 16) Heterologous ribonucleoprotein-A2 (hnRNP-A2) overexpression 17) L-plastin 18) Ganglioside fucosyl-GM1 19) Mob-1 expression (mob-1) (mob-1) ( Homology with proinflammatory cytokines).

[0187] In addition to markers for cell permanence known in the art, markers for cell permanence can also be identified using other methods known in the art. For example, cellular permanent markers can be identified by analyzing specific molecules (eg, nucleic acid molecules and polypeptide molecules) that are unique to specific permanent cell types.

II. Transgenic totipotent porcine cells Transforming the totipotent porcine cells of the present invention into transgenic totipotent cells that are concomitantly tolerable to those of ordinary skill in the art using readily available materials and methods. Can be. Once nuclear DNA has been modified in the totipotent cells of the invention, embryos and animals resulting from these cells can also contain the modified nuclear DNA.
Thus, transgenic and chimeric animals can be produced by applying to the totipotent cells of the present invention materials and methods readily available to the average technician in the art. For example, “Transgenic Animals Secreting Desired
EPO264166 entitled Proteins Into Milk, “Isolation of Componen
WO 94/19935, entitled “Method for Ide, entitled“ ts of Interest From Milk ”
WO93 / 2243 entitled "ntifying Transgenic Preimplantation Embryos"
2. WO95 / 1 entitled "Transgenic Production of Antibodies in Milk"
7085, Hammer et al., 1985, Nature 315: 680-685; Miller et al.
1986, J. Endocrinology 120: 481-488; Williams et al., 1992.
J. Ani. Sci. 70: 2207-2111; Piedrahita et al., 1998, Biol.
eprod. 58: 1321-1329; Piedrahita et al., 1997, J. Reprod. Fert.
(Supplement) 52: 245-254; and Nottle et al., 1997, J. Reprod. Fert.
(Supplement) 52: 245-254, each of which is incorporated herein by reference, including all diagrams, drawings and tables.

Methods for producing transgenic cells typically involve (1) assembling a suitable DNA construct useful for inserting the specific DNA sequence into the nuclear genome of the cell;
Transfecting the A construct into cells and (3) allowing random insertion and / or homologous recombination to take place. Modifications resulting from this process can be the insertion of the appropriate DNA construct (s) into the target genome, deletion of DNA from the target genome, and / or mutation of the target genome.

DNA constructs include genes of interest and various elements, such as regulated promoters, insulators, enhancers and repressors, and DNs.
An element for ribosome binding to RNA transcribed from the A construct may be included. The DNA construct may also encode a ribozyme and antisense DNA and / or RNA identified herein above. These examples are familiar to the average person skilled in the art and are not meant to be limiting.

Effective recombinant DNA that can be used in conjunction with DNA sequences for regulatory elements
Because of the genes readily available in the technology and database and commercial sectors, the average technician in the art can easily generate DNA constructs suitable for the establishment of transgenic cells using the materials and methods described herein. I can do it.

Transfection techniques are well known to those of ordinary skill in the art, and materials and methods for performing transfection of a DNA construct into cells are commercially available. For example, materials that can be used to transfect cells with a DNA construct include lipophilic compounds, such as Lipofectin ™
, Superfect ™, LipoTAXI ™ and CLON Fectin ™. By inducing a particular lipophilic compound, liposomes can be formed to mediate transfection of the DNA construct into cells. In addition, cells can be transfected with nucleic acid molecules by using cation-based transfection agents known in the art (eg, calcium phosphate precipitation). Also, nucleic acid molecules can be transferred to cells by utilizing electroporation techniques known in the art. Further, exogenous DNA can be introduced into cells by using particle bombardment techniques known in the art. Target sequences from a DNA construct can be inserted into specific regions of the nuclear genome by rational design of the DNA construct. These design techniques and methods are familiar to the average engineer in the art. U.S. Patent 5,633,067, "Method of Producing a Transgenic Bovin
e or Transgenic Bovine Embryo ", DeBoer et al., May 27, 1997, US Patent 5,612,205," Homologous Recombination in Mammalian Cells ", Kay.
Et al., March 18, 1997, and PCT Publication WO 93/22432, "Meth
od for Identifying Transgenic Pre-Implantation Embryos ", each of which is incorporated herein by reference, including all schemes, drawings and tables.
When the NA sequence is inserted into the nuclear genome of a cell, the location of the insertion region and the frequency of insertion of the target DNA sequence into the nuclear genome can be confirmed by methods familiar to those skilled in the art.

Once a transgene or transgenes have been inserted into the nuclear genome of a totipotent cell, the cells can be used as a nuclear donor to clone transgenic animals. Descriptions of specific examples for transgenic animals are described in more detail below.

A. Diseases and Parasites Insertion of a desired DNA sequence into the nuclear DNA of a cell can increase the resistance of a cloned transgenic animal to a particular parasite, disease and source of infection. Examples of parasites include parasites, flies, mites and fleas. Examples of infection sources include bacteria, fungi and viruses. Examples of diseases include atrophic rhinitis, cholera, leptospirosis, pseudorabies, and brucellosis. These examples are not limiting and the invention encompasses any disease or parasite or source of infection known in the art. For example, Hagan & Bruners Infectious Diseases of Domestic Animals (
7th Edition), Gillespie & Timoney, Copyright 1981 (Cornell University Press, Ithaca, New York).

A transgene may reduce resistance to a particular parasite or disease by completely eliminating or partially reducing the symptoms of the disease or parasite condition, or by producing proteins that control the parasite or disease. May be granted.

B. DNA Construct Elements and Production of DNA Constructs Depending on the nature of the host, a wide variety of transcriptional and translational regulatory sequences may be used. The transcriptional and translational regulatory signals can be derived from a viral source, such as adenovirus, bovine papillomavirus, cytomegalovirus, simian virus, etc.
Regulatory signals are associated with specific gene sequences that have a high level of expression potential. Alternatively, promoters from mammalian expression products such as actin, casein, alpha-lactalbumin, uroplakin, collagen, myosin and the like can be used. Transcriptional regulatory signals can be selected that suppress or activate such that expression of the gene product can be modulated. Of interest are regulatory signals that can be suppressed or initiated by external factors, such as chemicals or drugs. These examples are not limiting and the invention encompasses all regulatory elements. Other examples of regulatory elements are described herein.

C. Examples of Preferred Recombinant Products Various proteins and polypeptides can be encoded by genes contained within a DNA construct suitable for making transgenic cells. These proteins or polypeptides include hormones, growth factors, enzymes, coagulation factors, apolipoproteins, recipients, drugs, medicines, bioceuticals
), Functional foods, oncogenes, tumor antigens, tumor suppressors, cytokines, viral antigens, parasite antigens, bacterial antigens and chemically synthesized polymers and biosynthesis and / or modification by chemical, cellular and / or enzymatic processes There are polymers that have been used. Specific examples of these compounds include proinsulin, insulin, growth hormone, androgen recipient, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding protein, epidermal growth factor, TG
F-α, TGF-β, skin growth factor (PDGF), angiogenic factors (eg, acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin), angiogenesis inhibitors (eg, endostatin and Angiostatin), matrix proteins (type IV collagen, type VII collagen, laminin), oncogenes (ras, f
os, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p53 protein, cytokine recipient, IL-1, I
L-6, IL-8, IL-2, α, β or γ IFN, GMCSF, GCSF,
There are viral capsid proteins and proteins from viruses, bacteria and parasites. Other specific proteins or polypeptides that can be expressed include:
Phenylalanine hydroxylase, α-1-antitrypsin, cholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDL recipient, scavenger recipient for oxidized lipoprotein, each There are molecular variants, VEGF and combinations thereof. Other examples are monoclonal antibodies, antibody fragments, clotting factors, apolipoproteins, drugs, tumor antigens, viral antigens, parasite antigens, monoclonal antibodies and bacterial antigens. It is easy for one skilled in the art to understand that these proteins belong to a wide variety of protein types and that other proteins within or outside of these types can also be used. Should be admitted. These are merely examples and are not meant to be limiting in any case.

Also, the genetic material integrated into the cell from the DNA construct includes (1) a nucleic acid sequence not normally present in the target cell, and (2) a nucleic acid normally present in the target cell but not expressed at significant physiological levels. A molecule, (3) a nucleic acid sequence normally present in the target cell and normally expressed at a physiological target level, (4) another nucleic acid sequence that can be modified for expression in the target cell, and (5) any of the above. It should be noted that such combinations are included.

Furthermore, the DNA construct can be integrated into the nuclear DNA of the cell, where the integrated DN
A can be transcribed into a ribonucleic acid molecule and cleave other RNA molecules at specific regions. RN
A ribonucleic acid molecule capable of cleaving A molecule is referred to in the art as a ribozyme. Ribozymes are themselves RNA molecules. Ribozymes can bind to discrete regions in an RNA molecule and then specifically cleave regions within or adjacent to the binding region. Thereby, ribozyme technology can reduce the amount of polypeptide translated from a previously intact message RNA molecule.

In addition, the DNA construct can be incorporated into the nuclear DNA of a cell to produce RNA that can bind to both a transcription-specific RNA or a DNA sequence. Nucleic acid sequences can be utilized in antisense technology to bind to messages (mRNA) and block translation of these messages. Thereby, antisense technology may block or partially block the synthesis of a particular polypeptide in a cell.

III. Nuclear transfer Nuclear transfer (NT) technology using non-totipotent cells is well known to the average technician in the art. For example, McGrath & Solter, May 12, 1987, "Nu
clear Transplantation in the Mammalian Embryo by Microsurgery and Cell F
usion "U.S. Patent No. 4,640,097; U.S. Patent No. 4,994,384 (Prather et al.);
And 5057420 (Massey et al.), Each of which is incorporated herein by reference, including all schematics, tables and drawings. Embodiments demonstrate NT technology that can effectively produce totipotent porcine embryos.

A. Nuclear Donor The totipotent cells of the present invention can be used as a nuclear donor in the NT method for generating cloned embryos. As described above, totipotent cells can be generated from almost any type of cell. In the case of NT technology, donor cells can be isolated from a growing cell mass, isolated from primary cell culture, or isolated from a cell line. The whole cell can be placed in the perivitelline space of the recipient oocyte, or the nuclear donor is aspirated into a needle and the needle is placed into the recipient oocyte;
The nuclear donor can be released and injected directly into the recipient oocyte by removing the needle without significantly disrupting the plasma membrane of the oocyte. Also, nuclei (eg, nucleoplasm) can be isolated from a nuclear donor and placed into the perivitelline space of a recipient oocyte, or injected, for example, directly into the recipient oocyte.

B. Recipient oocyte A recipient oocyte is typically an oocyte from which a portion of its egg quality has been removed, wherein the removed egg quality contains an oocyte nucleus. Enucleation techniques are familiar to the average technician in the art. For example, Nagashima et al., 1997, Mol.R.
eprod. Dev. 48: 339-343, Nagashima et al., 1992, J. Reprod. Dev.
8: 37-78, Prather et al., 1989, Biol. Reprod. 41: 414-418, P.
rather et al., 1990, J. Exp. 255: 355-358; Saito et al., 199
2, Assis. Reprod. Tech. Andro. 259: 257-266, and Terlouw et al.
992, Theriogenology 37: 309, each of which is hereby incorporated by reference, including all schematics, tables and drawings.

Oocytes can be isolated from the oviducts and / or ovaries of living animals by the salping or transvaginal oocyte harvesting methods known in the art and described herein. obtain. In addition, oocytes can be removed from dead animals. For example,
Ovaries can be obtained from slaughterhouses and oocytes can be aspirated from these ovaries. Oocytes can also be isolated from the ovaries of a freshly killed animal or when the ovaries are frozen and / or thawed.

[0205] Oocytes can be matured in a variety of media familiar to the average technician in the art. One example of such a medium suitable for oocyte maturation is described in the embodiment below. Oocytes can mature well in this type of medium at 39 ° C. in an environment containing 5% CO ₂ . Oocytes can be cryopreserved and then thawed before placing the oocytes in the maturation medium. Cryopreservation methods for cells and embryos are known in the art, as discussed herein.

[0206] Components of the oocyte maturation medium can include molecules that inhibit oocyte maturation. Examples of such components are 6-dimethylaminopurine (DMAP) and isobutylmethylxanthine (IBMX). Although IBMX has been reported to reversibly block oocytes, its retention maintenance efficiency is quite low. For example, Rose-Hellkant and Ba
vister, 1996, Mol. Reprod. Develop. 44: 241-249. However, oocytes can be arrested at the germinal vesicle stage with relatively high efficiency by incubating the oocytes in an effective concentration of IBMX at 31 ° C. Preferably, the oocytes are incubated for the entire time they are being collected. Suitable concentrations of IBMX for oocyte maturation are 0.01 mM to 20 mM IBMX, preferably 0.05 mM to 10 mM IBMX, and more preferably about 0.1 mM IBMX to about 0.5 mM. IBMX, and most preferably 0.1.
1 mmol IBMX to 0.5 mmol IBMX.

[0207] The nuclear donor cells and the recipient oocytes can be from the same or different species. For example, totipotent porcine cells can be inserted into porcine enucleated oocytes. Alternatively, totipotent boar cells can be inserted into domesticated pig oocytes. Any nuclear donor / recipient oocyte combination is encompassed by the present invention. Preferred is
Nuclear donor and recipient oocytes from the same species. Cross-spec
ies) By utilizing NT technology, endangered or extinct cloned animals can be produced.

[0208] Oocytes can be activated by electrical and / or non-electrical means before, during, and / or after a nuclear donor is introduced into a recipient oocyte. For example, an oocyte can be placed in a medium containing one or more components suitable for non-electrical activation prior to fusion with a nuclear donor. Alternatively, the cybrids can be placed in a medium containing one or more components suitable for non-electrical activation. The activation process is discussed in considerable detail below.

C. Injection / fusion Fusion nuclear donors can be transferred to oocytes using a variety of materials and methods familiar to those of ordinary skill in the art. In one example, a nuclear donor can be injected directly into a recipient oocyte. This direct injection involves gradually withdrawing the nuclear donor into the needle, puncturing the recipient oocyte with the needle, releasing the nuclear donor into the oocyte, and removing the needle from the oocyte without significantly disrupting its membrane. Can be achieved. Suitable needles may be formed from glass capillaries, as specified in the art and by the publications cited herein.

In another example, at least a portion of the plasma membrane from a nuclear donor and recipient oocyte may be fused together by utilizing techniques familiar to the average artisan in the art. Willadsen, 1986, Nature 320: 63-65.
Reference, including all schemes, tables and drawings, is hereby incorporated by reference. Typically, lipid membranes can be fused together by electrical and chemical means, as specified above and in other publications incorporated herein by reference.

In an example of a non-electrical means of cell fusion, the cybrids are incubated in a solution containing polyethylene glycol (PEG) and / or Sendai virus. A wide range of molecular weight PEG molecules can be utilized for cell fusion.

[0212] Fusion processes not specifically discussed herein can be measured without undue experimentation. For example, modifications to cell fusion techniques can be monitored for their efficiency by observing the degree of cell fusion under a microscope. Then
The resulting embryo can be cloned and identified as a totipotent embryo by the same methods described herein above for identifying totipotent cells, which can include a selectable marker test and / or an animal developmental test.

D. Activation Oocytes and cybrid activation methods are known to the average technician in the art. US Patent 5,496,720, "Parthenogenic Oocyte Activation"
Susko-Parrish et al., Dated March 5, 1996, which is incorporated herein by reference, including all schematics, tables and drawings.

Electrical and non-electrical methods can be used to activate cells (eg, oocytes and cybrids). Although the use of non-electrical activation means is not necessary, non-electrical activation may increase the development potential of the cybrids, especially when young oocytes are used as recipients.

Examples of electrical techniques for cell activation are well known in the art. Published April 23, 1998, Piedraheidra and Blazer, WO 98/16630, which is hereby incorporated by reference, including all schematics, tables and drawings, and U.S. Pat.
See 994384 and 5057420. Non-electrical cell activation means may include any method known in the art for increasing the probability of cell division. Examples of non-electrical means for activating a nuclear donor and / or recipient cells ethanol, inositol triphosphate (IP _3), Ca ²⁺ ionophore and protein kinase inhibitors, such as 6-dimethylamino-purine, temperature change , Protein synthesis inhibitors (eg,
Cyclohexamide), phorbol esters such as phorbol 12-myristate 13-acetate (PMA), mechanical technology, thapsigarg
in) and by introducing into the semen factor. Semen factors may include all components of semen that increase the probability of cell division. Other non-electrical activation methods include cryogenic stimulation and / or mechanical stress of one or more cells.

Examples of preferred protein kinase inhibitors are protein kinase A, G and C inhibitors such as 6-dimethylaminopurine (DMAP), staurosporine,
2-aminopurine, sphingosine. Tyrosine kinase inhibitors may also be used to activate cells.

Activating materials and methods not specifically discussed herein may be ascertained by modifying the specific conditions specified below and in the exemplary protocol described in US Pat. No. 5,496,720. .

The activation efficiency and totipotency obtained by modifying the activation method can be identified by the methods described above in the section entitled “Identifying Totipotent Cells”. To identify a totipotent embryo,
One or more tests, such as (a) identifying specific markers for totipotent cells in the embryo, and (b) testing whether the embryo is totipotent by developing the embryo into an animal May be included. Accordingly, modifications to the activation methods described herein cannot be explicitly described herein, but the invention encompasses all of these modifications.

F. Manipulation of embryos obtained from nuclear transfer Embryos obtained from the NT process can be manipulated in various ways. The present invention relates to a cloned embryo obtained from at least one NT. Embodiments of the present invention include:
It has been demonstrated that two or more NT methods can increase the production efficiency of totipotent embryos. Embodiments have shown that the incorporation of two or more NT treatments into the method of producing clonal totipotent embryos can promote placental development. Further, by increasing the number of NT cycles included in the method for producing totipotent embryos,
The factors necessary to convert a non-totipotent cell to a totipotent cell can be indicated. The effect of two or more NT cycle incorporations on the totipotency of generated embryos is a surprising result and has not been previously identified or investigated in the art.

Incorporating two or more NT cycles into the cloned totipotent embryo method may provide yet another advantage. Numerous NTs to establish clone totipotent embryos
Incorporating treatments provides a way to increase the number of clonal totipotent embryos.

When multiple NT treatments are used to form clonal totipotent embryos, oocytes matured for a period of time can be used as recipients in the first, second or subsequent NT treatment. For example, if the first NT and the second NT are performed, the first NT may utilize an oocyte matured for about 53 hours as a recipient, and the second NT may be aged for less than about 53 hours. The allowed oocytes can be used as recipients. Alternatively, the first NT may utilize an oocyte aged about 53 hours as a recipient, and the second NT may use an oocyte aged more than about 53 hours for a two-cycle NT method recipe. Can be used as an entry. Furthermore, in both NT cycles,
Oocytes aged about 53 hours can be used as recipients, oocytes aged less than about 53 hours in both NT cycles can be used as recipients, and both NT cycles can be used as recipients from about 53 hours. Oocytes matured for a long time can be used as recipients in the two-cycle NT method.

In the case of NT technology that incorporates two or more NT cycles, one or more NT cycles may be preceded, followed, and / or performed simultaneously.
As previously described herein, the activation step may be performed by electrical and / or non-electrical means as described herein. The embodiments described below describe an NT technique that incorporates an activation step after one NT cycle. However,
The activation step may also be performed at the same time as the NT cycle (eg, simultaneously with the NT cycle), and / or the activation step may be performed before the NT cycle. Clonal totipotent embryos obtained from the NT cycle can be (1) isolated or (2) further developed as is.

When embryos are separated, the separated embryo-derived cells can be used to establish cultured cells. Either type of embryonic cells can be used to establish cultured cells. These cultured cells are sometimes referred to in the scientific literature as embryonic stem cells or embryonic stem-like cells. Embryonic stem cells can be derived from early, morula and blastocyst stage embryos. Numerous methods for the production of cultured embryonic cells are known to the average technician in the art. These methods are listed in the specific references cited herein above.

When an embryo is developed into a fetus in utero, cells isolated from the developing fetus can be used to establish cultured cells. In a preferred embodiment, primordial germ cells, germ ridge cells and fetal fibroblasts can be isolated from such fetuses. Cultured cells having the specific morphology described herein can be referred to as embryonic germ cells (EG cells). These cultured cells can be established by using culture methods familiar to the average person skilled in the art. Such methods are listed in the publications previously cited herein and are discussed herein.

The clonal totipotent embryo obtained from NT can also be manipulated by cryopreserving and / or thawing the embryo. For example, Nagashima et al., 1989, Japanese J. An
im.Reprod. 35: 130-134 and Feng et al., 1991, Theriogenology 3.
5: 199, each of which is hereby incorporated by reference, including all tables, diagrams and drawings. Other embryo manipulation methods include in vitro culture methods, performing embryo transfer to maternal recipients, separating blastomeres for the NT process, blastomeres or inner cell mass to establish cell lines used in the NT method. There are cell separation, embryo division, embryo aggregation, embryo sex determination and embryo biopsy. The specific procedures are not meant to be limiting, and the present invention is intended to include all embryo manipulation procedures known to those of ordinary skill in the art.

IV. Development of cloned embryos Identification of Totipotent Embryos Totipotent embryos can be identified by the method described in the section entitled "Identifying Totipotent Cells." Individual cells can be separated and subjected to similar tests. Testing involves, for example, confirming the presence or absence of a marker. Also, a totipotent embryo can be identified by developing the embryo until it passes the first trimester of pregnancy, or preferably, develops into a live born animal. Also described herein are methods for identifying markers for totipotency.

B. In Vitro Culture of Embryos The cloning method discussed here offers the advantage of culturing cells and embryos in vitro before transplantation into female recipients. Methods for in vitro culturing of embryos are well known to those skilled in the art. For example, Nagashima et al., 1997, M
ol. Reprod. Dev. 48: 339-343, Petters & Wells, 1993, J. Am. Reprod
.Fert. (Supplement) 48: 61-73, Reed et al., 1992, Theriogenology 37: 9.
5-109, and Dobrinsky et al., 1996, Biol. Reprod. 55: 1069-1.
074, each of which is hereby incorporated by reference, including all schemes, tables and drawings. Further, embodiments relating to a medium suitable for in vitro culture of cloned embryos are described below. The feeder layer may or may not be used for in vitro culture of cloned embryos. Feeder cells are described above and in the embodiments below.

C. In utero growth of embryos Clonal embryos can be cultured in an artificial or natural uterine environment after NT treatment and the embryo in vitro culture process. Examples of artificial development environments have been developed, some of which are known to those skilled in the art. The components of the artificial environment can be modified, for example, by altering the amount of one or more components and by monitoring the growth rate of the embryo.

[0229] Methods for transferring embryos into the uterus of animals are also well known in the art, as discussed above. Preferably, the stage of development of the embryo (s) correlates with the estrous cycle of the animal.

[0230] Embryos from one species can be placed into the uterine environment of an animal from another. For example, it has been shown in the art that bovine embryos can grow in ovine oviducts. Stic
e & Keefer, 1993, "Multiple generational bovine embryo cloning", B
iology of Reproduction 48: 715-719. The invention includes all combinations of porcine embryos in any other ungulate uterine environment. Cross-sp
ecies) In utero growth can effectively produce endangered species of cloned animals. For example, a boar embryo can grow in the uterus of a domestic sow.

Once the embryo is placed in the uterus of a female recipient, the embryo can grow to the date of delivery. Alternatively, the embryos can grow in situ and then be removed at selected times. Surgical methods of removing a fetus from the womb before birth are well known in the art.

V. Pig Cloned Animals As previously described herein, the present invention provides the advantage that the phenotype of an animal can be assessed prior to cloning the animal. A variety of products can be isolated from cloned animals. For example, semen may be collected from an animal, such as a domestic boar. Semen can be cryopreserved. Semen can also be separated into sex-specific fractions of semen. Spaulding, dated August 8, 1995, September 13, 1994 and June 4, 1991, respectively, entitled "Sex-associated Membrane Proteins and Method".
s for Increasing the Probability that Offspring Will be of a Desired Sex
Nos. 5,439,362, 5,346,990 and 502,244, each of which is hereby incorporated by reference, including schemes, drawings and tables. As discussed above, the method of collecting semen is described in US Pat. Well-known to the average engineer in the industry.

In addition, the present invention relates, in part, to products collected from porcine cloned animals. These products may be body fluids or organs separated from animals, or products separated from body fluids or organs. In a preferred embodiment, the product, eg, meat, can be collected from a swine cloned animal. In another aspect, the present invention relates to the procedure of determining the phenotype of a swine, a castrated animal, and then cloning the cloned animal so that it has a regenerative function and can be used for semen production. Things. Another preferred embodiment of the present invention relates to products such as xenograft material, semen, embryos, oocytes, all types of cells and progeny taken from the cloned animal of the present invention.

The xenograft material described herein above may include all cellular material extracted from one organism and transferred to another organism. Medical methods for extracting cellular material from one organism and transplanting it into another organism are well known to the average technician in the art. Examples of preferred xenograft cell material may be selected from the group consisting of liver, lung, heart, nerve, gall bladder and pancreatic cell material.

As discussed in the previous section, transgenic animals can be generated from the methods of the invention by using transgenic techniques familiar to the average technician in the art. Preferably, porcine cloned transgenic animals are produced by these methods. The clonal transgenic animals can be genetically engineered so that they are resistant or partially resistant to their unique disease or parasite. Examples of these diseases and parasites are outlined in the previous section.

Further, such that the cloned transgenic animal produces a recombinant product,
They can be genetically engineered. Examples of recombinant products are outlined in the previous section. Expression of these products can be directed to particular cells or regions in the cloned transgenic animal by selectively manipulating promoter and regulatory elements appropriate for achieving this goal.

For example, human recombinant products can be expressed in cattle urine by operably linking a uroplakin promoter to a DNA sequence encoding the recombinant product. Alternatively, examples of the selective expression of human recombinant products in the milk of bovine animals are familiar to the average skilled artisan.

Once the recombinant product or products have been expressed in a particular tissue or body fluid of the cloned transgenic animal, the appropriate tissue or body fluid can be harvested using methods known in the art. Recombinant products can be purified from body fluids or tissues by using standard purification techniques familiar to those skilled in the art.

EXAMPLES The following examples are non-limiting examples of various aspects and features of the present invention and are merely representative.

Embodiment 1 Preparation of Feeder Cell Layer A feeder cell layer of fetal fibroblasts was prepared from mouse fetuses on days 10 to 20 of gestation. The head, liver, heart, and gastrointestinal tract were removed, the remaining organs were washed, and incubated at 37 ° C. in 0.053% trypsin and 0.53 mM EDTA (Gibco BRL cat # 15400-096). Loose cells were penicillin, streptomycin, 10% fetal calf serum, and 0.1 mM 2-
Cultured in tissue culture dishes containing MEM-alpha supplemented with mercaptoethanol. Feeder cell cultures were established at 37.4 ° C., 3.5% CO ₂ , and humidified air over 2 to 3 weeks or more. Before being used as feeder cells, mouse fibroblasts were pretreated with mitomycin C (Caobiocem cat # 47589) for 3 hours to a final concentration of 10 μg / ml and pre-equilibrated growth medium was added. Before washing 5 times with PBS. Feeder cells are established from fetal pigs as described below to establish cultured fetal fetal fibroblasts.

Example 2 Establishment of Cultured Nuclear Donor Cells from Tissues Other than Embryos One of the advantages provided by the materials and methods identified herein is that virtually any type of progenitor cell Can also establish totipotent cells.

[0242] 1. Establishment of Cultured Cells from Fetal Bovine Tissues Germline raised cells were isolated from fetal bovine and degraded by proteolytic enzymes (pronase E or trypsin). EG formed directly from the reproductive ridge cells
Cells having the cell morphology are digested, but the mouse feeder cell layer (Example 1)
See also) Grow faster when established on top. Addition of growth factors, hrLIF and bFGF to the cell culture medium has little effect on the establishment and proliferation of EG cells. The growth rate of EG cells increased when 17 mM D-glucose was injected into the culture medium. Cell culture medium contained alpha-MEM, 10% fetal calf serum, and 0.1 mM 2-mercaptoethanol.

Fetuses at 33, 36, 47, and 54 days of age were used as progenitor progenitor progenitor cells. EG cells from 33, 36, and 47 day old fetuses were much more difficult to establish and generally grew slowly. When cell cultures are established from germ-lift cells isolated from 33, 36, and 47-day-old fetuses, EG cells are overgrown by other cell types, typically elongated cells such as fibroblasts. Grew up. In contrast, EG cells from 54 day old fetuses grew relatively quickly and formed large colonies within 1-2 weeks. EG cells are physically isolated from other competing cell types and are monolayers of apparently one cell type with typical EG cell morphology, the morphology described herein. As they grow up to confluence.

[0244] 2. Establishment of Cultured Porcine Cumulus Cells Cumulus cells were isolated from an in vitro matured porcine cumulus-oocyte complex. Ovary-derived cumulus-oocyte complex was recovered from the slaughterhouse and matured in porcine maturation medium (described below). Cumulus-oocyte complex is placed in a 15 ml centrifuge tube at 500
Cumulus cells were stripped from the cumulus-oocyte complex by placing with μL TL HEPES and agitating the contents using a vortex mixer (mixing at half speed for 3 minutes).

The cell suspension formed from the stirred cumulus-oocyte complex is 10 mL TL
After washing twice with HEPES, they were transferred to Petri dishes. Petri dishes were placed under a microscope and oocytes were manually removed from the cell suspension using a small-bore pipette.

Once the oocytes were removed from the cell suspension, the remaining cumulus cells were placed in a 15 mL centrifuge tube. The tube was placed in a centrifuge and pelleted into cumulus cells. The pelleted cumulus cells were placed in a 60 mm tissue culture dish with ME.
In a 5 mL culture medium containing M-alpha, 10% fetal calf serum, 0.1 mM β-mercaptoethanol and an aliquot, approximately 25,000 cells /
Resuspended at a cell density of mL. Cumulus cells were cultured using basic cell culture techniques. Cell culture medium is 3
Changed every 4 days, cumulus cells were passaged when they became confluent.

[0247] 3. Establishment of cultured porcine amniotic fluid cells Amniotic fluid is collected from pregnant female pigs using the guided needle technique. The amniotic fluid is transferred into a 15 mL conical tube and centrifuged at 1000 × g for 10 minutes. The supernatant fluid is removed leaving 1 mL of fluid on the cell pellet. The cell pellet is resuspended in the 1 mL amniotic fluid. A 0.5 mL amniotic fluid cell sample was placed in a T25 culture flask with 4.5 mL of complete AminoMax medium (90 mL of AminoMax-C100 Basal medium + 15 mL of AminoMax-C100 supplement [Gi
bco BRL]) and placed in an incubator with air containing 5% CO ₂ at a temperature of 39 ° C.

The amniotic fluid cells attach to the culture flask within about 4 days. Amniotic fluid cell cultures typically became 70% confluent within 7-14 days. Cells are passaged by removing the cell culture medium from the flask and washing the cells twice with EBSS medium or PBS medium (without calcium or magnesium, Gibco BRL).
0.2 ml of trypsin / EDTA solution (Gibco BRL) is added to the culture flask and incubated at 39 ° C. until cells are released from the culture flask surface. After the cells were released from the culture flask surface, cell culture media was added to the flask to inactivate trypsin and the cells were distributed to three new T25 flasks.

[0249] 4. Establishment of Cultured Porcine Fetal Fibroblasts Cultured porcine fetal fibroblasts were prepared from porcine fetuses between days 33 and 54 of gestation. The head, liver, heart, and digestive tract are removed, the remaining organs are washed, and C
a ²⁺ and Mg ²⁺ free Dulbecco's PBS (Gibco BRL Cat. No. 14190
-151) diluted with 0.05% trypsin and 0.53 mM EDTA (Gibco
BRL catalog number 15400-096) at 37 ° C. Loose cells were penicillin, streptomycin, 10% fetal calf serum (Hyclon
e Catalog number SH3007.03) and 0.1 mM 2-mercaptoethanol (Gib
Co BRL catalog number 21985-023) was cultured in tissue culture dishes containing MEM-alpha (Gibco BRL catalog number 32561-037) supplemented. Porcine fetal cells were established at 37.4 ° C., 3.5% CO ₂ , and humidified air over 2 to 3 weeks or more.

[0250] 5. Establishment of Cultured Porcine Fibroblasts from Mature Animals The first step to establishing totipotent cells from the tissue of an adult animal is to establish a primary culture of the isolated cells. Subsequent methods relate to ear punch samples, but the method is applicable to cells isolated from any type of tissue. The steps described below are preferably performed using aseptic methods. 1. Each ear sample is washed twice with 2 mL of trypsin / EDTA solution in two separate 35 mm Petri dishes. Each ear sample is processed separately. 2mL
Cut ear samples with sterile scissors and scalpel in 35 mm Petri dishes containing trypsin / EDTA solution from Finely cut pieces are 1m in diameter
m or less. 2. The minced ear pieces are placed in a trypsin / EDTA solution for 40-50 minutes.
Incubate with occasional swirling in a 37 ° C. incubator. Trypsin / EDTA solution is described in detail below. Dish may be wrapped in order to reduce the accumulation of CO ₂ in the stretchable material, such as Parafilm (registered). 3. Transfer the digested ear sample pieces to a 15 mL sterile tube. Wash dishes to collect digested ear slices with 2 mL of trypsin / EDTA solution;
Transfer the wash solution to the sterile tube. 4. Stir the tube at high speed for 2 minutes. Add 5.5 mL of cell culture medium (defined below) to inactivate the trypsin. 6. Centrifuge the 15 mL tube at 280 × g for 10 minutes. 7. Tilt the vessel to remove the supernatant and resuspend the cell debris in the remaining solution by tapping the side of the tube. 8. Add 2 mL of cell culture medium to the tube, then centrifuge as indicated in step 6. 9. Tilt the vessel to remove the supernatant and resuspend the cell debris as shown in step (7), then add 2 mL of culture. 10. Leave -8 in a 2 mL tube, leaving 2-3 ear samples for DNA analysis.
Store at 0 ° C. 11. Transfer the resuspended cells into a 35 mm media dish and wet 5% CO ₂ /
Incubate at 37 ° C. in 95% air. 12. Change media every 2-4 days.

[0251] Trypsin / EDTA solution was prepared by adding 0.05% trypsin (w / v) solution.
Trypsin-EDTA solution (Gibco
BRL catalog number 15400-096) was mixed with Ca ²⁺ -free and Mg ²⁺ -free Dulbecco's phosphate buffered saline (PBS) (Gibco BRL catalog number 14190-151). The solution was sterilized by filtering through a 0.2 μm filter.

The culture medium for cell culture is the minimum essential MEM-alpha (Gibco BRL Cat # 32561
-037) 10% fetal calf serum (Hyclone # SH30070.03), 0.1 mM 2-mercaptoethanol (Gibco BRL Cat. No. 21985-023), 100 U / mL penicillin, 100 μg / mL streptomycin, 0.25 μg / mL am It was prepared by mixing Hotel Shin B (Fungizone).

Example 3 Program Modification of Cultured Porcine Cells If the cultured cells established by the method taught in Example 2 are not clearly totipotent, the cells can be reprogrammed by the following method. Germ raised cell suspensions (final concentration 100,000 cells / ml) were placed in 35 mm Petri dishes containing a feeder layer of mouse early embryo fibroblasts.
The culture medium is MEM-alpha (Gibco BRL Cat. No. 32561-037), 10% fetal bovine serum (Hyclone Cat. No. SH30070.03) at 37.5 ° C.
, 0.1 mM 2-mercaptoethanol (Gibco BRL Cat. No. 21985-023
), 100 ng / ml human recombinant leukemia inhibitory factor (hrLIF; R & D System Catalog No. 250-L), 100 ng / ml bovine basic fibroblast growth factor (bFGF;
D System catalog number 133-FB) and 5% CO ₂ . Exogenous stem cell growth factors (eg, membrane-bound and soluble stem cell growth factors) need not be supplemented to the medium.

Culture medium supplemented with 100 ng / mL hrLIF and 100 ng / mL bFGF is incubated with the progenitor cells for typically 10 to 14 days. The supplemented medium is replaced every 2-4 days with fresh supplemented medium. At any time in this culture method, EG cells can be observed. These EG cells can be isolated from cell culture and cultured separately to establish a homogeneous EG cell culture.

To maintain the EG cell line, a dense population of cells is passaged weekly at a dilution of 1: 4 to 1: 8. The cells are passaged by incubating the cells with a 0.05% trypsin-0.53 mM EDTA mixture and preparing for a fresh culture in fresh growth medium. Enhance the ability of MEM-alpha medium to promote growth of MEM-alpha medium against totipotent cells by adding insulin-transferrin, sodium selenite medium additive (Sigma Cat # 11884) to the cell culture medium at a 1: 100 dilution. It is possible. Short-term cultivation using tyrosine tartrate (Sigma Catalog No. T3151) is performed as a preventative measure against mycoplasma contamination. Prior to NT, the cell line must contain DNA specific for the mycoplasma sequence.
The presence of mycoplasma by PCR performed using primers (Stratagene catalog number 302007) was examined.

Example 4: Oocyte maturation Aspiration and collection of oocytes The ovaries of sows were transported to the laboratory at 25 ° C. and immediately washed with 0.9% saline. A 3-6 mm follicle was vacuum aspirated using an 18 gauge needle and a 50 mL tube connected to a vacuum aspiration system where vacuum was regulated at a flow rate of 45 mL / min. After aspiration, the oocytes were allowed to settle for 5-10 minutes. Follicular fluid (pFF
) Was aspirated and, if necessary, set aside for use in the mature system.

Oocytes were resuspended in 20 mL Hepes-PVA medium by
After washing, the oocytes were allowed to settle. The medium is removed and the washing step is
Repeated more than once. Hepes-PVA medium contains 114 mM NaCl, 3 m
M KCl, 2 mM CaCl₂, 0.5 mM MgCl₂, 2 mM NaHCO ₃ , 0.3 mM NaH₂PO₄, 10 mM HEPES, 12 mM sorbitol
, 0.2 mM sodium pyruvate, 0.05 μL / mL Gentamici
, 0.07 g / L penicillin G, 0.1 g / L polyvinyl alcohol, and
And 2 mL / L sodium lactate at a pH of 7.4. Washed oocytes
The vesicles were then placed in a maturation medium.

[0258] 2. In vitro maturation Oocytes are washed three times in maturation medium and 50-20 oocytes / well are transferred to 0.5 ml hormone-containing maturation medium in 4-well plates for 20-22 hours. Was done. In addition, 6-8 follicle shell pieces are added to the wells, the addition of which is additional. These follicular shell pieces are known to improve the cytoplasmic maturation of porcine oocytes, which results in increased oocyte developmental potential. For example, Abeydeere et al., 1998, Biol. Reprod.
58: 213-218.

The maturation medium was 109 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ ,
1 mM MgSO ₄ , 25 mM NaHCO ₃ , 1 mM KH ₂ PO ₄ , 6 mM
It contains glucose, 1 mM glutamine, 12 mM sorbitol, 5 mg / L insulin, 100 IU / L penicillin G, and 50 mg / L streptomycin and has a pH of 7.4. Porcine follicular fluid (pFF) is about 10% (v / v
) Can be added to the maturation medium. Β-mercaptoethanol is 5
It can be added to the maturation medium to a final concentration of 0 μM. In addition, cysteine can be added to the maturation medium to a final concentration of 0.6 mM. The mature medium containing hormones is 10
ng / mL epidermal growth factor, 10 U / mL equine chorionic gonadotropin, 10 U / mL human chorionic gonadotropin, and 1 mM db-cAMP
Was contained.

The oocytes and follicle shell pieces were then transferred to 0.5 mL of fresh maturation medium,
After an additional 20-22 hours of maturation, the maturation medium here did not contain hormones or db-cAMP. Oocytes are 39% in 5.0% CO ₂ air
Matured at 40 ° C for a total of 40-44 hours.

Example 5 Nuclear Transfer 42-46 hours after maturation, oocytes were detached from cumulus cells by agitating mature oocytes for about 2 minutes. After agitation, the oocytes were evaluated for polar body formation rates. The polar body formation rate can be evaluated by visual inspection using a stereo microscope. It is possible to enhance the visual assessment of the rate of polar body formation by staining the oocytes in a medium containing 3 μg / mL Hoechst 33442 and 7.5 μg / mL cytochalasin B for about 20 minutes. The oocytes were then enucleated with an enucleation pipette.

After the oocytes were enucleated, the NT method was performed. In each NT method, the nuclear donor cells were adapted to the oocytes such that the portion of the cell membrane for each cell in contact with each other and the nuclear donor matched the oocyte transparencies. Porcine EG cells, cultured porcine germ cells, cultured porcine fibroblasts, cultured porcine cumulus cells, and cumulus cells isolated from the cumulus-oocyte complex after 48 hours of maturation are nuclear donors for the NT method. Was used as

After the nuclear donor was adapted to the enucleated oocyte, the two cells were fused. Three media can be used to fuse the cells. : (1) 0.25
M sorbitol, 0.1 mM CaOAc, 0.1 mM MgOAc, and 0
. 1% BSA; (2) 0.3 M mannitol, 0.1 mM MgSO ₄ , 0
. 5 mM CaCl ₂ and 0.1% BSA; (3) 0.3 M mannitol, 5% TL Hepes, and 0.3% BSA; and (4) Zimmerman medium. After the fusion, the fused oocytes contained 109 mM NaCl, 5 mM KCl,
2 mM CaCl ₂ , 1 mM MgSO ₄ , 25 mM NaHCO ₃ , 1 mM
KH ₂ PO ₄ , 6 mM glucose, 1 mM glutamine, 7 mM taurine,
They were placed in a culture containing 5 mM hypotaurine, 0.4% BSA, 100 IU / L penicillin G, and 50 mg / L streptomycin at pH 7.4 for 4 hours. The fused oocytes were also in CR2 medium (C amino acid supplemented).
R1 medium), the latter being described in US Pat. No. 5,096,822.
No. 3, "Bovine embryo medium", Rosenkrans Jr. et al., November 3, 1992, which is hereby incorporated by reference in its entirety, including all figures, tables, and descriptions.

Example 6: Activation After incubation for 4 hours after fusion, fused oocytes are incubated for 8 minutes in a ZI solution containing 10 μM ionomycin (TL Hepes stock + 1 mg / ml BSA), Z30 solution (TL Hepes stock +
Rinsed in 30 mg / ml BSA) for 8 minutes. The TL Hepes stock contains 114 mM NaCl, 3 mM KCl, 2 mM NaHCO ₃ , 0.3 mM
NaH ₂ PO ₄ , 10 mM Hepes, 2 mM CaCl ₂ , 0.5 mM
It contained MgCl ₂ , 2 ml / L sodium lactate, and 1 ml / L phenol red and had a pH of 7.4.

Oocytes were washed three times with TL Hepes PVA and then washed with C with BSA
The cells were placed in a 2 mM DMAP solution of R2 medium at 39 ° C. in 5.0% CO ₂ air for 4 hours. Subsequently, activation occurs, and the activated oocytes are transformed into TL Hepes
Rinsed three times with PVA and placed in porcine fetal culture medium.

Example 7: In Vitro Culture of Activated Oocytes and Embryos After removal of the DMAP solution, the activated oocytes and cytoplasmic hybrids were treated with TL Hep
es Washed three times in PVA and placed in porcine embryo culture medium for 4 days. The culture medium for embryo culture was 109 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ , 1 m
The pH was 7.4 containing M MgSO ₄ , 25 mM NaHCO ₃ , 1 mM KH ₂ PO ₄ , 6 mM glucose, 1 mM glutamine, 12 mM sorbitol, 5 mg / L insulin, 100 IU / L penicillin G, and 50 mg / L streptomycin. Activated oocytes and cytoplasmic hybrids also
It is also possible to culture in R2 medium. On the fourth day, the embryos are cracked and transferred to a fresh embryo culture medium supplemented with 10% fetal calf serum (v / v) for an additional 3 days. On day 7, the fetus grows to the blastocyst.

Embodiment 8 FIG. Recloning Method One or more additional recloning cycles can be performed to increase the totipotency of the nuclear donor cells. In the recloning cycle, embryos from the first NT cycle were treated with pronase E (1-3 mg / mL in TL HEPES) after treatment with cytochalasin B when the embryos were in the morula stage.
) Or mechanically. A blastomere is placed in the perivitelline space of the enucleated oocyte (52-54 hours after maturation as taught in Example 4).

One blastomere was fused to enucleated oocytes by electrofusion in a 500 μm chamber with an electric pulse of 105 V for 15 μs in an isotonic sorbitol solution (0.25 M) at 30 ° C. Is done. It is not required that the fused oocytes undergo chemical activation for the recloning cycle.

After the oocyte fusion, the fused oocytes from the second NT cycle were at 39 ° C.
And cultured in an embryo culture medium under humidified air containing 5% CO ₂ . Such an embryo can be transferred to a female recipient.

Example 9: Pig Embryo Transfer to Maternal Recipients and Development of Cloned Pigs Ten pigs were injected with gonadotropin (PG600®), resulting in approximately 2-5 recipients Showed estrus for 3-5 days after treatment. Synchronized pigs are utilized for embryo transfer either on the third or fifth day of estrus. Early embryos (<8 cell stage) were implanted into recipients on day 3 of estrus, and late embryos (> 8 cell stage) were implanted separately into recipients on day 5 of estrus. Animals are anesthetized, a ventral midline incision is made, and the reproductive organs are exposed. The embryo transfer procedure consists of transferring the deposited 30-50 early embryos into the fallopian tubes, or transferring the late embryos (morula and / or blastocyst stage embryos) into the uterine horn. The recipient
It is examined whether pregnancy has begun and is continuously monitored by weak ultrasonography.

Example 10 Cloning of Transgenic Pigs Transgenic cells suitable for establishing transgenic pig clones can be prepared from cells isolated from adult animals. FIG. 3 illustrates a method that can be used to establish such transgenic cells. While using the teachings of the present invention, transgenic cells can be established from almost any type of cell, FIG. 3 shows a method for establishing transgenic embryonic undifferentiated cells and transgenic totipotent cells. Is illustrated.

[0272] Fibroblast cultures can be established from ear punches extracted from pigs as described above. In addition, cultured fibroblasts can be established from pig fetuses. Individual cells are isolated from this cell culture and can be used as nuclear donors in nuclear transfer methods. One nuclear transfer cycle or multiple nuclear transfer cycles can be applied. Other additional steps are shown in the previous embodiment.

The cells can then be transduced with the DNA construct. Cells can be transduced in multiple steps as shown in FIG. Materials and methods for preparing transgenic cells are provided in the publications cited above. The totipotent cell according to the present invention is a DN comprising: (a) an antibiotic resistance gene; (b) a DNA sequence encoding a protein or a group of proteins; and (c) a promoter element or a group of elements.
A can be transduced. The transduced cells are selected for genetic modification by selecting a cell culture medium containing the antibiotic. The transgenic cells are then screened for transgenic modifications using one or more screening techniques. Examples of these techniques include: (1) polymerase chain reaction, (
2) Southern blotting and (3) fiber-FISH method. These techniques are well known to those of ordinary skill in the art. The latter two techniques are available to examine the number of copies of the gene sequence inserted into the nuclear DNA of embryonic germ cells.

These screening methods are applicable to the transduced cells in any of the steps shown in FIG. Transgenic animal clones are established by utilizing transgenic cells as nuclear donors in the NT method as shown in Example 5. The fused oocytes generated by such a method are used in the methods taught in Examples 6, 7, and 9, and additionally 8 for the generation of transgenic porcine clones.

While the invention has been described and illustrated in sufficient detail to enable those skilled in the art to make and use the invention, it will be apparent that variations, modifications and improvements can be made without departing from the spirit and scope of the invention. It is.

Those skilled in the art will readily appreciate that the present invention may be well adapted to carry out the objects and obtain the essential objects and advantages, as well as the stated objects and advantages. Cell lines, embryos, animals and procedures and methods for making them are set forth and illustrated by the preferred examples, and are not intended to limit the scope of the invention. These modifications fall within the spirit of the invention and are defined by the appended claims. It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are set forth at the level of ordinary skill in the art to which this invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each and every publication was specifically and individually indicated and incorporated herein by reference. It has been incorporated.

The invention illustratively described herein may be practiced without element or a plurality of elements, limitations, or limitations not specifically disclosed herein. Thus, for example, in any case herein, any term "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. Terms and phrases that have been used are used as words of description rather than of limitation, and the use of such words or phrases is not limited to the feature shown or described, or to some of the features While not intending to exclude anything, it is recognized that various modifications are possible within the scope of the claims of the present invention. Thus, while the present invention has been specifically disclosed by preferred embodiments and additional features, modifications and variations of the disclosure herein may be employed by those skilled in the art, It should be understood that various changes and modifications are considered to be within the scope of the present invention as defined in the appended claims.

Further, where features or aspects of the present invention are described with reference to Markush-type groups, one of ordinary skill in the art will appreciate that the present invention may be applied to any individual member of a Markush-type group, or any member of a subgroup. Is also described by its Markush-type group. For example, if X is indicated as being selected from the group consisting of bromine, chlorine, and iodine, then X
Claims wherein X is bromine and claims wherein X is bromine and chlorine will be fully described. Other embodiments are set forth in the following claims.

[Brief description of the drawings]

FIG. 1 illustrates aspects of the invention relating to generating totipotent cells from progenitor cells. This figure shows that totipotent cells can be generated from embryonic stem cells, primordial germ cells, and cells isolated from animals. The source of progenitor cells shown in FIG. 1 is not limiting, and other sources of progenitor cells are described herein.

FIG. 2 illustrates aspects of the invention relating to totipotent cell cell lines and routes for establishing clonal animals. Fibroblasts can be isolated from any of the sources described herein.

FIG. 3 illustrates aspects of the invention for establishing a cloned transgenic cell line and a cloned transgenic animal.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 ) Inventor Eric Jay Forsberg 53707 Niagara Court, Fitchburg, Wisconsin, United States (72) Inventor Michael Di Bishop 53, 964 Rio, Wisconsin, United States Hall No. 4628 F-term (reference) 4B024 AA01 AA05 AA07 CA01 DA02 GA18 GA30 HA20 4B065 AA90X AA90Y AB01 AC20 BA08 BB15 BB34 BC41 BD41 BD44 CA41 CA44 CA60

Claims (42)

[Claims] 1. A totipotent porcine cell, wherein the totipotent cell has been cultured. 2. The totipotent porcine cell according to claim 1, wherein the cell has an embryonic germ cell morphology. 3. A system comprising: (a) isolating at least one progenitor cell; (b) culturing the progenitor cell in a cell culture medium; and (c) introducing a stimulating factor into the progenitor cell. A totipotent porcine cell produced by a method comprising the step of converting to a porcine porcine cell. 4. A method comprising the steps of: a) isolating one or more progenitor cells, and (b) establishing totipotent porcine cells by culturing the progenitor cells in a cell culture medium. A totipotent porcine cell having an embryonic germ cell morphology. 5. The stimulating factor comprises a recipient ligand cocktail.
The described totipotent porcine cells. 6. The method according to claim 1, wherein the progenitor cells are derived from non-embryonic cells, differentiated cells, fibroblasts, fetal cells, fetal fibroblasts, primordial germ cells, germ cells, embryonic cells, cumulus cells, amniotic cells, and hepatocytes. The totipotent porcine cell according to claim 3 or 4, which is selected from the group consisting of: 7. The method of claim 1, wherein the porcine cells comprise modified nuclear DNA.
The described totipotent porcine cells. 8. The totipotent porcine cell of claim 1, 2, 3, or 4, wherein the porcine cell does not appreciably stain for alkaline phosphatase. 9. The totipotent porcine cell of claim 1, wherein the porcine cell has been manipulated. 10. The totipotent porcine cell according to claim 3, wherein the cell culture medium comprises a feeder cell. 11. The totipotent porcine cell of claim 3 or 4, wherein the cell culture medium contains a significant concentration of glucose. 12. The totipotent porcine cell of claim 11, wherein the substantial concentration of glucose is greater than 10 millimolar glucose. 13. The totipotent porcine cell of claim 12, wherein the substantial concentration of glucose is about 17 mM glucose. 14. The totipotent porcine cell of claim 3, wherein the cell culture medium comprises one or more feeder cells. 15. A system comprising: (a) isolating at least one progenitor cell; (b) culturing the progenitor cell in a cell culture medium; and (c) introducing a stimulating factor into the progenitor cell. A method for producing a totipotent porcine cell, comprising the step of converting the cell into a porcine cell. 16. A totipotent porcine cell comprising the steps of: a) isolating one or more progenitor cells and (b) establishing the totipotent porcine cells by culturing the progenitor cells in a cell culture medium. The method of producing, wherein the totipotent pig cells have an embryonic germ cell morphology. 17. A cloned porcine embryo, wherein the embryo is totipotent and the embryo originates from a totipotent cell. 18. The embryo is totipotent and is produced by a method comprising nuclear transfer between a nuclear donor and an oocyte, wherein the nuclear donor is a totipotent porcine cell and the oocyte is capable of forming an embryo. A cloned porcine embryo at the stage of obtaining. 19. The method according to claim 19, wherein the nuclear transfer comprises activating totipotent porcine cells and oocytes.
A cloned porcine embryo according to claim 18. 20. (a) establishing a nuclear transfer oocyte by transferring a nuclear donor to an oocyte; and (b) establishing a porcine embryo by non-electrical activation of the nuclear transfer oocyte. A cloned porcine embryo produced by a method comprising: 21. The cloned porcine embryo of claim 17, 18 or 20, wherein one or more cells of the embryo comprises modified nuclear DNA. 22. The cloned porcine embryo of claim 18 or 20, wherein the oocyte is derived from a species different from the species of the nuclear donor. 23. The cloned porcine embryo of claim 20, wherein the transposition comprises a fusion step of a nuclear donor and an oocyte. 24. The non-electrical activation comprises: (a) increasing intracellular levels of divalent cations in the oocyte, and (b) reducing phosphorylation of cellular proteins in the oocyte. 21. The cloned porcine embryo of claim 20. 25. The cloned porcine embryo of claim 20, wherein the non-electrical activation comprises introducing ionomycin and DMAP into a nuclear transfer embryo. 26. The cloned porcine embryo of claim 23, wherein ionomycin is about 10 micromolar and DMAP is about 2 millimolar. 27. The nuclear donor is an embryonic germ cell and embryonic stem cell, cumulus cell, amniotic cell, non-embryonic cell, differentiated cell, fibroblast, fetal cell, fetal fibroblast, primordial germ cell, genital bulge 21. The cloned porcine embryo of claim 20, which is selected from the group consisting of cells, embryonic cells, differentiated cells, undifferentiated cells, and hepatocytes. 28. The cloned porcine embryo of claim 17, 18, or 20, wherein the embryo is manipulated. 29. The cloned porcine embryo of claim 28, wherein the operation comprises culturing the cloned porcine embryo in vitro. 30. The cloned porcine embryo of claim 28, wherein the manipulating comprises transferring the cloned porcine embryo into the uterus of a suitable ungulate. 31. The operation comprises: (a) separating the cloned porcine embryo into one or more individual cells; and (b) (i) individual cells of (a); and (ii) an oocyte. 29. The cloned porcine embryo of claim 28, comprising performing at least one subsequent nuclear transfer between cells. 32. A method of producing a cloned porcine embryo, comprising nuclear transfer between a nuclear donor and an oocyte, wherein the nuclear donor is a totipotent porcine cell and the oocyte is in a stage capable of embryogenesis. Method. 33. (a) establishing a nuclear transfer oocyte by transferring a nuclear donor to an oocyte; and (b) establishing a porcine embryo by non-electrical activation of the nuclear transfer oocyte. A method for producing a cloned porcine embryo, comprising: 34. The method of claim 17, 18, 19, 20, 21, 22, 23, 24.
, 25, 26, 27, 28, 29, 30, and 31. A porcine cloned animal derived from the embryo of any one of the above. 35. (a) Claims 17, 18, 19, 20, 21, 22, 23
Producing a cloned porcine embryo according to any one of claims 24, 25, 26, 27, 28, 29, 30 and 31; and (b) manipulating the cloned porcine embryo to develop into an animal Porcine cloned animal produced by a method comprising the step of adding. 36. (a) Establishing a nuclear transfer oocyte by transferring a nuclear donor to an oocyte; (b) Establishing a cloned porcine embryo by non-electrical activation of a nuclear transfer oocyte And (c) transferring the porcine embryo to a female recipient, where the porcine embryo develops into a porcine clonal animal. 37. The one or more cells of the animal comprising modified nuclear DNA.
A porcine cloned animal according to claim 34, 35 or 36. 38. A method comprising the steps of: (a) isolating at least one component from a porcine animal; and (b) transferring the component to another animal, wherein said component is from the group consisting of bodily fluids, cells, tissues and organs. The method of use of the porcine cloned animal to be selected. 39. The method of claim 38, wherein the bodily fluid is semen. 40. The method of claim 38, wherein the organ is selected from the group consisting of neural tissue, brain tissue, spleen, heart, lung, gallbladder, pancreas, testis, ovary, and kidney. 41. A pig comprising the steps of (a) producing a cloned porcine embryo by the method of claim 32 or 33, and (b) manipulating the cloned porcine embryo to develop into an animal. A method for producing a cloned animal. 42. (a) Establishing a nuclear transfer oocyte by transferring a nuclear donor to an oocyte; (b) Establishing a cloned porcine embryo by non-electrical activation of the nuclear transfer oocyte And (c) transferring the porcine embryo to a female recipient, where the porcine embryo develops into a porcine clonal animal.